Method for generating immune cells resistant to arginine and/or tryptophan depleted microenvironment

ABSTRACT

The present invention pertains to engineered immune cells, method for their preparation and their use as medicament, particularly for immunotherapy. The engineered immune cells of the present invention are characterized in that at least one gene selected from a gene encoding GCN2 and a gene encoding PRDM1 is inactivated or repressed. Such modified Immune cells are resistant to an arginine and/or tryptophan depleted microenvironment caused by, e.g., tumor cells, which makes the immune cells of the invention particularly suitable for immunotherapy. The invention opens the way to standard and affordable adoptive immunotherapy strategies using immune cells for treating different types of malignancies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage entry under 35 U.S.C. § 371 ofInternational Application No. PCT/EP2015/057865, filed Apr. 10, 2015,which claims priority to Danish Patent Application No. PA201470209,filed Apr. 11, 2014. The disclosure of the prior applications are herebyincorporated in their entirety by reference.

FIELD OF THE INVENTION

The present invention pertains to engineered immune cells, such asT-cells, method for their preparation and their use as medicament,particularly for immunotherapy. The engineered immune cells of thepresent invention are characterized in that at least one gene selectedfrom a gene encoding GCN2 (general control nonderepressible 2; alsoknown as eukaryotic translation initiation factor 2 alpha kinase 4,EIFF2AK4) and a gene encoding PRDM1 (PR domain containing 1, with ZNFdomain; also known as B lymphocyte-induced maturation protein 1,BLIMP-1) is inactivated or repressed. Such modified immune cells areresistant to an arginine and/or tryptophan depleted microenvironmentcaused by, e.g., tumor cells, which makes the immune cells of theinvention particularly suitable for immunotherapy. The invention opensthe way to standard and affordable adoptive immunotherapy strategiesusing immune cells for treating different types of malignancies.

BACKGROUND OF THE INVENTION

Cellular adaptive immunity is mediated by T-lymphocytes, also known asT-cells, which upon recognition of a non-self or tumoral antigen caneither destroy the target cell or orchestrate an immune response withother cells of the immune system.

Adoptive immunotherapy, which involves the transfer of autologousantigen-specific T cells generated ex vivo, is a promising strategy totreat viral infections and cancer. The T-cells used for adoptiveimmunotherapy can be generated either by expansion of antigen-specific Tcells or redirection of T-cells through genetic engineering (Park,Rosenberg et al. 2011). Transfer of viral antigen specific T-cells is awell-established procedure used for the treatment of transplantassociated viral infections and rare viral-related malignancies.Similarly, isolation and transfer of tumor specific T-cells have beenshown to be successful in treating melanoma.

Novel specificities in T-cells have been successfully generated throughthe genetic transfer of transgenic T cell receptors or chimeric antigenreceptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptorsconsisting of a targeting moiety that is associated with one or moresignaling domains in a single fusion molecule. In general, the bindingmoiety of a CAR consists of an antigen-binding domain of a single-chainantibody (scFv), comprising the light and variable fragments of amonoclonal antibody joined by a flexible linker. Binding moieties basedon receptor or ligand domains have also been used successfully. Thesignaling domains for first generation CARs are derived from thecytoplasmic region of the CD3zeta or the Fc receptor gamma chains. Firstgeneration CARs have been shown to successfully redirect T cellcytotoxicity, however, they failed to provide prolonged expansion andanti-tumor activity in vivo. Signaling domains from co-stimulatorymolecules including CD28, OX-40 (CD134), and 4-1BB (CD137) have beenadded alone (second generation) or in combination (third generation) toenhance survival and increase proliferation of CAR modified T-cells.CARs have successfully allowed T-cells to be redirected against antigensexpressed at the surface of tumor cells from various malignanciesincluding lymphomas and solid tumors (Jena, Dotti et al. 2010).

While it is thus possible to redirect T-cell cytotoxicity towards tumorcells, these later cells may still dampen the immune response by escapemechanisms. One such escape mechanism is the elimination of certainamino acids such as arginine and tryptophan from their localmicroenvironment by production of arginase and Indoleamine2,3-dioxygenase (IDO1).

Most reports have associated arginase activity with the need formalignant cells to produce polyamines to sustain their rapidproliferation. However, arginase tends to inhibit T-cell proliferationand activation.

Rodriguez et al. (2004) found that L-arginine (L-Arg) plays a centralrole in several biologic systems including the regulation of T-cellfunction. L-Arg depletion by myeloid-derived suppressor cells producingarginase I is seen in patients with cancer inducing T-cell anergy. Theyshowed that L-Arg starvation could regulate T-cell-cycle progressioninsofar as T cells cultured in the absence of L-Arg are arrested in theG0-G1phase of the cell cycle. This was associated with an inability of Tcells to up-regulate cyclin D3 and cyclin-dependent kinase 4 (cdk4).Silencing of cyclin D3 reproduced the cell cycle arrest caused by L-Argstarvation. They also found that Signaling through GCN2 kinase wastriggered during amino acid starvation.

A recent study demonstrated that arginase is expressed and released fromLeukemia blasts and is present at high concentrations in the plasma ofpatients with acute myeloid leukemia (AML), resulting in suppression ofT-cell proliferation (Mussai, F. et al. 2013). The study showed that theimmunosuppressive activity of AML blasts can be modulated through smallmolecule inhibitors of arginase and inducible nitric oxide synthase,strongly supporting the hypothesis that AML creates an immunosuppressivemicroenvironment that contributes to the pancytopenia observed atdiagnosis. High arginase activity has been also described in patientswith solid tumors, in particular in gastric, colon, breast, and lungcancers, and more particularly in small cell lung carcinoma (Suer etal., 1999). It is also considered that the following reaction catalyzedby arginase+H2O→urea+ornithine increases urea and ornithineconcentration is the environment of tumors, which may have a negativeimpact on lymphocytes. On another hand, the inhibition of arginase invivo was found to decrease tumor growth in mice as per the study byRodriguez et al. (2004).

The metabolic enzyme IDO1 contributes to the balance between toleranceversus inflammation in a number of experimental models. Expression ofIDO1 in APCs, such as macrophages and dendritic cells, can suppress Tcell responses as observed during mammalian pregnancy, inflammatoryconditions, autoimmunity and tumor resistance. IDO1 was found to beover-expressed by plasmacytoid dendritic cells in tumor draining lymphnodes (Munn, D. H. et al., 2004) as well as in child acute myeloidleukemia (AML) (Rutella, S. et al., 2013) and patients with chroniclymphocytic leukemia (Lindström V., et al. 2013). IDO1 catabolizes theessential amino acid tryptophan, thus decreasing concentrations in thelocal microenvironment as well as generating biologically activedownstream metabolites. Studies in both yeast and mice revealed thatGCN2 also plays a role in the response to tryptophan deprivation. PRDM1(also referred to as BLIMP-1) is a protein, which expression levelparallels that of IDO1, and that is up-regulated in situation oftryptophan deprivation.

It thus appears that production of arginase and/or IDO1, through aminoacid deprivation, represents a significant component of tumor escape,which needs to be addressed by innovative immunotherapy strategies,especially those involving T-cells.

SUMMARY OF THE INVENTION

The above need is addressed, according to the present invention, byrepressing or disrupting GCN2 and/or PRDM1 protein formation in immunecells, such as T-cells, to make them resistant to arginine and/ortryptophan depletion. Through the experiments shown in the presentspecification, GCN2 and PRDM1 proteins are found to act as sensors ofarginine and/or tryptophan starvation, which can be switched off toavoid anergy of immune cells, particularly T-cells, withoutsignificantly dampening their activity. The resulting immune cellsremain in condition to proliferate in the local microenvironment ofarginase producing cells, and thus are prompt to confer an improvedimmune response against tumors.

According to one aspect, the present invention provides a method forpreparing an engineered immune cell, in particular an engineered T cell,comprising:

modifying an immune cell, such as a T-cell, by inactivating orrepressing a gene encoding GCN2 (such as human GCN2 or a functionalvariant thereof) and/or a gene encoding PRDM1 (such as human PRDM1 or afunctional variant thereof).

According to certain embodiments, the immune cell is modified byinactivating a gene encoding GCN2 (e.g., the human GCN2 gene; NCBIReference Sequence: NG_034053.1). The inactivation of the GCN2 gene may,for instance, be achieved by genome modification, more particularlythrough the expression in the immune cell of a rare-cutting endonucleaseable to selectively inactivate said gene by DNA cleavage, preferablydouble-strand break. Such rare-cutting endonuclease may be aTALE-nuclease, meganuclease, zinc-finger nuclease (ZFN), or RNA guidedendonuclease.

According to particular embodiments, the immune cell is a human immunecell which is modified by inactivating a gene encoding human GCN2 as setforth in SEQ ID NO: 1 (NCBI Reference Sequence: NP_001013725.2) or afunctional variant thereof which has at least about 80%, such as atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98% or at least about 99%,sequence identity with the human GCN2 set forth in SEQ ID NO: 1 over theentire length of SEQ ID NO: 1.

According to certain embodiments, the immune cell is modified byrepressing a gene encoding GCN2 (e.g., the human GCN2 gene; NCBIReference Sequence: NG_034053.1).

According to certain other embodiments, the immune cell is modified byinactivating a gene encoding PRDM1 (e.g., the human PRDM1 gene; NCBIReference Sequence: NG_029115.1). The inactivation of the PRDM1 genemay, for instance, be achieved by genome modification, more particularlythrough the expression in the immune cell of a rare-cutting endonucleaseable to selectively inactivate said gene by DNA cleavage, preferablydouble-strand break. Such rare-cutting endonuclease may be aTALE-nuclease, meganuclease, zinc-finger nuclease (ZFN), or RNA guidedendonuclease.

According to another embodiment, said rare-cutting endonuclease is a DNAguided endonuclease. As an example, such endonuclease may be theArgonaute proteins (Ago). Ago proteins from bacteria such as Thermusthermophilus (strain HB27) have been recently described in bacteria toact as a barrier for the uptake and propogation of foreign DNA (SwartsD. C, et al. Nature 507: 258-261) In vivo, Tt Ago is loaded with 5′phosphorylated DNA guides, from 13 to 25 base pairs that are mostlyplasmid derived and have a strong bias for a 5′-end deoxycytidine. Thesesmall interfering DNAs guide TtAgo cleave complementary DNA strands athigh temperature (75° C.). WO2014189628A (Caribou biosciences) disclosessuch complex comprising an Argonaute and a designed nucleicacid-targeting nucleic acid.

According to particular embodiments, the immune cell is a human immunecell which is modified by inactivating a gene encoding human PRDM1 asset forth in SEQ ID NO: 2 (NCBI Reference Sequence: NP_001189.2) or afunctional variant thereof which has at least about 80%, such as atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98% or at least about 99%,sequence identity with human PRDM1 as set forth in SEQ ID NO: 2 over theentire length of SEQ ID NO: 2.

According to certain other embodiments, the immune cell is modified byrepressing a gene encoding PRDM1 (e.g., the human PRDM1 gene; NCBIReference Sequence: NG_029115.1).

According to certain other embodiments, the immune cell is modified byinactivating both the gene encoding GCN2 (e.g., the human GCN2 gene) andthe gene encoding PRDM1 (e.g., the human PRDM1 gene). The inactivationof the GCN2 gene and PRDM1 gene may, for instance, be achieved by genomemodification, more particularly through the expression in the immunecell of rare-cutting endonucleases able to selectively inactivate saidgenes by DNA cleavage, preferably double-strand break. Such rare-cuttingendonucleases may independently be a TALE-nuclease, meganuclease,zinc-finger nuclease (ZFN), or RNA guided endonuclease.

According to particular embodiments, the immune cell may be furtherengineered to make it non-alloreactive, especially by inactivating oneor more genes involved in self-recognition, such as those, for instance,encoding components of T-cell receptors (TCR). This can be achieved by agenome modification, more particularly through the expression in theimmune cell, particular T-cell, of a rare-cutting endonuclease able toselectively inactivate by DNA cleavage, preferably double-strand break,at least one gene encoding a component of the T-Cell receptor (TCR),such as the gene encoding TCR alpha or TCR beta. Such rare-cuttingendonuclease may be a TALE-nuclease, meganuclease, zing-finger nuclease(ZFN), or RNA guided endonuclease. Preferably, the rare-cuttingendonuclease is able to selectively inactivate by DNA cleavage the genecoding for TCR alpha.

According to optional embodiments, the immune cell may be furtherengineered to express a Chimeric Antigen Receptor (CAR) directed againstat least one antigen expressed at the surface of a malignant cell.Particularly, said CAR is directed against an antigen commonly expressedat the surface of solid tumor cells, such as 5T4, ROR1 and EGFRvIII.Said CAR may also be directed against an antigen commonly expressed atthe surface of liquid tumors, such as CD123, or CD19.

The present invention thus provides in a further aspect engineeredimmune cells, in particular isolated engineered immune cells,characterized in that a gene encoding GCN2 and/or a gene encoding PRDM1is inactivated or repressed.

According to certain embodiments, an engineered immune cell, inparticular isolated engineered immune cell, is provided wherein a geneencoding GCN2 (e.g., the human GCN2 gene) is inactivated.

According to certain other embodiments, an engineered immune cell, inparticular isolated engineered immune cell, is provided wherein a geneencoding GCN2 (e.g., the human GCN2 gene) is repressed.

According to certain other embodiments, an engineered immune cell, inparticular isolated engineered immune cell, is provided wherein a geneencoding PRDM1 (e.g., the human PRDM1 gene) is inactivated.

According to certain other embodiments, an engineered immune cell, inparticular isolated engineered immune cell, is provided wherein a geneencoding PRDM1 (e.g., the human PRDM1 gene) is repressed.

According to certain other embodiments, an engineered immune cell, inparticular isolated engineered immune cell, is provided wherein both agene encoding GCN2 (e.g., the human GCN2 gene) and a gene encoding PRDM1(e.g., the human PRDM1 gene) are inactivated.

According to certain other embodiments, an engineered immune cell, inparticular isolated engineered immune cell, is provided wherein both agene encoding GCN2 (e.g., the human GCN2 gene) and a gene encoding PRDM1(e.g., the human PRDM1 gene) are repressed.

According to certain other embodiments, an engineered immune cell, inparticular isolated engineered immune cell, is provided wherein a geneencoding GCN2 (e.g., the human GCN2 gene) is inactivated and a geneencoding PRDM1 (e.g., the human PRDM1 gene) is repressed.

According to certain other embodiments, an engineered immune cell, inparticular isolated engineered immune cell, is provided wherein a geneencoding GCN2 (e.g., the human GCN2 gene) is repressed and a geneencoding PRDM1 (e.g., the human PRDM1 gene) is inactivated.

According to certain embodiments, an immune cell is provided whichexpresses a rare-cutting endonuclease able to selectively inactivate byDNA cleavage in said cell a gene encoding GCN2. More particularly, suchimmune cell comprises an exogenous nucleic acid molecule comprising anucleotide sequence encoding said rare-cutting endonuclease, which maybe a TALE-nuclease, meganuclease, zing-finger nuclease (ZFN), or RNAguided endonuclease.

According to particular embodiments, said rare-cutting endonucleasebinds to a sequence set forth in SEQ ID NO: 3. According to otherparticular embodiments, said rare-cutting endonuclease binds to asequence set forth in SEQ ID NO: 4.

According to certain other embodiments, an immune cell is provided whichexpresses a rare-cutting endonuclease able to selectively inactivate byDNA cleavage in said cell a gene encoding PRDM1. More particularly, suchimmune cell comprises an exogenous nucleic acid molecule comprising anucleotide sequence encoding said rare-cutting endonuclease, which maybe a TALE-nuclease, meganuclease, zing-finger nuclease (ZFN), or RNAguided endonuclease.

According to certain other embodiments, an immune cell is provided whichexpresses a rare-cutting endonuclease able to selectively inactivate byDNA cleavage in said cell a gene encoding GCN2 and a rare-cuttingendonuclease able to selectively inactivate by DNA cleavage in said cella gene encoding PRDM1. More particularly, such immune cell comprises oneor more exogenous nucleic acid molecules comprising one or nucleotidesequences encoding said rare-cutting endonucleases, which independentlymay be a TALE-nuclease, meganuclease, zing-finger nuclease (ZFN), or RNAguided endonuclease.

According to particular embodiments, the immune cell may further have atleast one inactivated gene encoding a component of the TCR receptor.More particularly, such immune cell may express a rare-cuttingendonuclease able to selectively inactivate by DNA cleavage, preferablydouble-strand break, said at least one gene encoding a component of theT-Cell receptor (TCR). Accordingly, said immune cell may comprise anexogenous nucleic acid molecule comprising a nucleotide sequence codingfor a rare-cutting endonuclease able to selectively inactivate by DNAcleavage at least one gene coding for one component of the T-Cellreceptor (TCR). The disruption of TCR provides a non-alloreactive immunecell that can be used in allogeneic treatment strategies.

According to optional embodiments, the immune cell may be engineered toexpress a Chimeric Antigen Receptor (CAR) directed against at least oneantigen expressed at the surface of a malignant cell. Particularly, theimmune cell comprises an exogenous nucleic acid molecule comprising anucleotide sequence encoding said CAR. According to particularembodiments, said CAR is directed against an antigen selected from CD19,CD33, CD123, CS1, BCMA, CD38, 5T4, ROR1 and EGFRvIII. The binding of thetarget antigen by the CAR has the effect of triggering an immuneresponse by the immune cell directed against the malignant cell, whichresults in degranulation of various cytokine and degradation enzymes inthe interspace between the cells.

As a result of the present invention, engineered immune cells can beused as therapeutic products, ideally as an “off the shelf” product, foruse in the treatment or prevention of medical conditions such as cancer.

Thus, the present invention further provides an engineered immune cellof the present invention or a composition, such as a pharmaceuticalcomposition, comprising same for use as a medicament. According tocertain embodiments, the engineered immune cell or composition is foruse in the treatment of a cancer, and more particularly for use in thetreatment of a solid or liquid tumor. According to particularembodiments, the engineered immune cell or composition is for use in thetreatment of a cancer selected from the group consisting of lung cancer,small lung cancer, breast cancer, uterine cancer, prostate cancer,kidney cancer, colon cancer, liver cancer, pancreatic cancer, and skincancer. According to other particular embodiments, the engineered immunecell or composition is for use in the treatment of a sarcoma. Accordingto other particular embodiments, the engineered immune cell orcomposition is for use in the treatment of a carcinoma. According tomore particular embodiments, the engineered immune cell or compositionis for use in the treatment of renal, lung or colon carcinoma. Accordingto other particular embodiments, the engineered immune cell orcomposition is for use in the treatment of leukemia, such as acutelymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chroniclymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), andchronic myelomonocystic leukemia (CMML). According to other particularembodiments, the engineered immune cell or composition is for use in thetreatment of lymphoma, such as Hodgkin's or Non-Hodgkin's lymphoma.According to certain embodiment, the engineered immune cell originatesfrom a patient, e.g. a human patient, to be treated. According tocertain other embodiment, the engineered immune cell originates from atleast one donor.

It is understood that the details given herein with respect to oneaspect of the invention also apply to any of the other aspects of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation of an engineered immune cell accordingto the invention expressing a rare-cutting endonuclease able toselectively inactivate by DNA cleavage a GCN2 encoding gene and/or arare-cutting endonuclease able to selectively inactivate by DNA cleavagea PRDM1 encoding gene.

FIG. 2: Measurement by flow cytometry of live cell concentration ofhuman T-cells transfected with mRNA encoding a TALE nuclease specificfor TRAC (KO TRAC) or untransfected human T-cells (WT; wild type) afterexposure for 72 hours at 37° C. to increasing concentrations ofrecombination arginase I (0.5-1500 ng/ml) in Xvivo15 medium complementedwith 5% human AB serum and 20 ng/ml human IL2 (100 μl per well in a96-well plate). The data confirm that both untransfected T-cells andT-cells treated with TRAC specific TALE nuclease are sensitive toarginine deprivation in vitro.

FIG. 3: Results of T7 endonuclease assay on genomic DNA isolated fromhuman T-cells transfected with mRNA encoding GCN2 specific TALEnucleases. The presence of lower molecular bands compared to samplesobtained from untransfected T-cells indicates cleavage activity of bothTALENs used.

FIG. 4: Measurement by flow cytometry of live cells concentration ofhuman T-cells transfected with mRNA encoding GCN2 specific TALEnucleases (KO1 and KO2) or untransfected human T-cells (WT, wild type)after incubation for 72 hours at 37° C. in RPMI1640 medium withincreasing concentrations of arginine added. The data show cells treatedwith GCN2 specific TALE nuclease survive better at lower concentrationsof arginine, and thus provides resistance to immunosuppression in atumor microenvironment where arginase is secreted.

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined herein, all technical and scientific termsused have the same meaning as commonly understood by a skilled artisanin the fields of gene therapy, biochemistry, genetics, and molecularbiology.

All methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,with suitable methods and materials being described herein. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willprevail. Further, the materials, methods, and examples are illustrativeonly and are not intended to be limiting, unless otherwise specified.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, CurrentProtocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley andson Inc, Library of Congress, USA); Molecular Cloning: A LaboratoryManual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, N.Y.:Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J.Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Harries & S. J. Higgins eds. 1984); TranscriptionAnd Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture OfAnimal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); ImmobilizedCells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide ToMolecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelsonand M. Simon, eds.-in-chief, Academic Press, Inc., New York),specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, “GeneExpression Technology” (D. Goeddel, ed.); Gene Transfer Vectors ForMammalian Cells (J. H. Miller and M. P. Cabs eds., 1987, Cold SpringHarbor Laboratory); Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

Methods for Preparing Engineered T-Cells

In a general aspect, the present invention pertains to methods forpreparing engineered immune cells, such as T-cells or natural killer(NK) cells.

Accordingly, the present invention provides a method for preparing anengineered immune cell comprising:

-   -   modifying an immune cell, such as a T-cell, by inactivating or        repressing a gene encoding GCN2 (such as human GCN2 or a        functional variant thereof) and/or a gene encoding PRDM1 (such        as human PRDM1 or a functional variant thereof).

According to certain embodiments, the immune cell is modified byinactivating a gene encoding GCN2 (e.g., the human GCN2 gene; NCBIReference Sequence: NG_034053.1). The inactivation of the GCN2 gene may,for instance, be achieved by genome modification, more particularlythrough the expression in the immune cell of a rare-cutting endonucleaseable to selectively inactivate said gene by DNA cleavage.

According to particular embodiments, the immune cell is a human immunecell which is modified by inactivating a gene encoding human GCN2 as setforth in SEQ ID NO: 1 (NCBI Reference Sequence: NP_001013725.2) or afunctional variant thereof which has at least about 80%, such as atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98% or at least about 99%,sequence identity with the human GCN2 set forth in SEQ ID NO: 1 over theentire length of SEQ ID NO: 1.

According to certain other embodiments, the immune cell is modified byinactivating a gene encoding PRDM1 (e.g., the human PRDM1 gene; NCBIReference Sequence: NG_029115.1). The inactivation of the PRDM1 genemay, for instance, be achieved by genome modification, more particularlythrough the expression in the immune cell of a rare-cutting endonucleaseable to selectively inactivate said gene by DNA cleavage.

According to particular embodiments, the immune cell is a human immunecell which is modified by inactivating a gene encoding human PRDM1 asset forth in SEQ ID NO: 2 (NCBI Reference Sequence: NP 001189.2) or afunctional variant thereof which has at least about 80%, such as atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98% or at least about 99%,sequence identity with human PRDM1 as set forth in SEQ ID NO: 2 over theentire length of SEQ ID NO: 2.

According to certain other embodiments, the immune cell is modified byinactivating both a gene encoding GCN2 (e.g., the human GCN2 gene) and agene encoding PRDM1 (e.g., the human PRDM1 gene). The inactivation ofthese genes may, for instance, be achieved by genome modification, moreparticularly through the expression in the immune cell of a rare-cuttingendonuclease able to selectively inactivate by DNA cleavage the geneencoding GCN2 and a rare-cutting endonuclease able to selectivelyinactivate by DNA cleavage the gene encoding PRDM1.

By “inactivating” or “inactivation of” a gene it is intended that thegene of interest (e.g. a gene encoding GCN2 or PRDM1) is not expressedin a functional protein form. In particular embodiments, the geneticmodification of the method relies on the expression, in provided cellsto engineer, of a rare-cutting endonuclease such that same catalyzescleavage in one targeted gene thereby inactivating said targeted gene.The nucleic acid strand breaks caused by the endonuclease are commonlyrepaired through the distinct mechanisms of homologous recombination ornon-homologous end joining (NHEJ). However, NHEJ is an imperfect repairprocess that often results in changes to the DNA sequence at the site ofthe cleavage. Mechanisms involve rejoining of what remains of the twoDNA ends through direct re-ligation (Critchlow and Jackson 1998) or viathe so-called microhomology-mediated end joining (Betts, Brenchley etal. 2003; Ma, Kim et al. 2003). Repair via non-homologous end joining(NHEJ) often results in small insertions or deletions and can be usedfor the creation of specific gene knockouts. Said modification may be asubstitution, deletion, or addition of at least one nucleotide. Cells inwhich a cleavage-induced mutagenesis event, i.e. a mutagenesis eventconsecutive to an NHEJ event, has occurred can be identified and/orselected by well-known method in the art.

A rare-cutting endonuclease to be used in accordance with the presentinvention to inactivate the gene encoding GCN2 may, for instance, be aTALE-nuclease, meganuclease, zinc-finger nuclease (ZFN), or RNA guidedendonuclease (such as Cas9).

According to a particular embodiment, the rare-cutting endonuclease is aTALE-nuclease.

According to another particular embodiment, the rate-cuttingendonuclease is a homing endonuclease, also known under the name ofmeganuclease.

According to another particular embodiment, the rare-cuttingendonuclease is a zinc-finger nuclease (ZNF).

According to another particular embodiment, the rare-cuttingendonuclease is a RNA guided endonuclease. According to a preferredembodiment, the RNA guided endonuclease is the Cas9/CRISPR complex.

In order to be expressed in the immune cell, a rare-cutting endonucleaseused in accordance with the present invention to inactivate a geneencoding GCN2 may be introduced into the cell by way of an exogenousnucleic acid molecule comprising a nucleotide sequence encoding saidrare-cutting endonuclease. Accordingly, the method of the presentinvention may comprise introducing into the immune cell an exogenousnucleic acid molecule comprising a nucleotide sequence encoding arare-cutting endonuclease able to selectively inactivate by DNA cleavagea gene encoding GCN2 (e.g., the human GCN2 gene). As a result, anengineered T-cell is obtained which expresses a rare-cuttingendonuclease able to selectively inactivate in said cell by DNA cleavagea gene encoding GCN2.

According to particular embodiments, the rare-cutting endonucleasetargets (e.g., binds to) a sequence set forth in SEQ ID NO: 3. Accordingto other particular embodiments, the rare-cutting endonuclease targets(e.g., binds to) a sequence set forth in SEQ ID NO: 4.

A rare-cutting endonuclease to be used in accordance with the presentinvention to inactivate the PRDM1 gene may, for instance, be aTALE-nuclease, meganuclease, zinc-finger nuclease (ZFN), or RNA guidedendonuclease (such as Cas9).

According to a particular embodiment, the rare-cutting endonuclease is aTALE-nuclease.

According to another particular embodiment, the rate-cuttingendonuclease is a homing endonuclease, also known under the name ofmeganuclease.

According to another particular embodiment, the rare-cuttingendonuclease is a zinc-finger nuclease (ZNF).

According to another particular embodiment, the rare-cuttingendonuclease is a RNA guided endonuclease. According to a preferredembodiment, the RNA guided endonuclease is the Cas9/CRISPR complex.

In order to be expressed in the T-cell, a rare-cutting endonuclease usedin accordance with the present invention to inactive the gene encodingPRDM1 may be introduced into the cell by way of an exogenous nucleicacid molecule comprising a nucleotide sequence encoding saidrare-cutting endonuclease. Accordingly, the method of the presentinvention may comprise introducing into the T-cell an exogenous nucleicacid molecule comprising a nucleotide sequence encoding a rare-cuttingendonuclease able to selectively inactivate by DNA cleavage the geneencoding PRDM1 (e.g., the human PRDM1 gene). As a result, an engineeredT-cell is obtained which expresses a rare-cutting endonuclease able toselectively inactivate by DNA cleavage the gene encoding PRDM1.

According to certain embodiments, the immune cell is modified byrepressing a gene encoding GCN2 (e.g., the human GCN2 gene; NCBIReference Sequence: NG_034053.1).

According to certain other embodiments, the immune cell is modified byrepressing a gene encoding PRDM1 (e.g., the human PRDM1 gene; NCBIReference Sequence: NG_029115.1).

By “repressing” or “repression of” a gene it is intended that theexpression of a gene of interest (e.g. a gene encoding GCN2 or PRDM1) ina modified cell is reduced compared to the expression of said gene in anunmodified cell of the same type. In particular, “repressing” or“repression of” a gene is meant that the expression of a gene ofinterest (e.g. a gene encoding GCN2 or PRDM1) in a modified cell isreduced by at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% or about 100% compared to the expression ofsaid gene in an unmodified cell of the same type.

Repression of a gene of interested can be achieved by any suitable meansknown in the art. For example, the expression of a gene of interest maybe reduced by gene silencing techniques such as the use of antisenseoligonucleotides, ribozymes or interfering RNA (RNAi) molecules, such asmicroRNA (miRNA), small interfering RNA (siRNA) or short hairpin RNA(shRNA).

It is also contemplated by the present invention that the engineeredimmune cell, in particular in case of an engineered T-cell, of thepresent invention does not express a functional T-cell receptor (TCR) onits cell surface. T-cell receptors are cell surface receptors thatparticipate in the activation of T-cells in response to the presentationof antigen. The TCR is generally made from two chains, alpha and beta,which assemble to form a heterodimer and associates with theCD3-transducing subunits to form the T-cell receptor complex present onthe cell surface. Each alpha and beta chain of the TCR consists of animmunoglobulin-like N-terminal variable (V) and constant (C) region, ahydrophobic transmembrane domain, and a short cytoplasmic region. As forimmunoglobulin molecules, the variable region of the alpha and betachains are generated by V(D)J recombination, creating a large diversityof antigen specificities within the population of T cells. However, incontrast to immunoglobulins that recognize intact antigen, T-cells areactivated by processed peptide fragments in association with an MHCmolecule, introducing an extra dimension to antigen recognition by Tcells, known as MHC restriction. Recognition of MHC disparities betweenthe donor and recipient through the T-cell receptor leads to T-cellproliferation and the potential development of graft versus host disease(GVHD). It has been shown that normal surface expression of the TCRdepends on the coordinated synthesis and assembly of all sevencomponents of the complex (Ashwell and Klusner 1990). The inactivationof TCR alpha or TCR beta can result in the elimination of the TCR fromthe surface of T-cells preventing recognition of alloantigen and thusGVHD. The inactivation of at least one gene coding for a TCR componentthus renders the engineered immune cell less alloreactive. By“inactivating” or “inactivation of” a gene it is meant that the gene ofinterest (e.g., at least one gene coding for a TCR component) is notexpressed in a functional protein form.

Therefore, the method of the present invention in accordance withparticular embodiments further comprises inactivating at least one geneencoding a component of the T-cell receptor. More particularly, theinactivation is achieved by using (e.g., introducing into the immunecell, such as T-cell) a rare-cutting endonuclease able to selectivelyinactivate by DNA cleavage, preferably double-strand break, at least onegene encoding a component of the T-cell receptor. According toparticular embodiments, the rare-cutting endonuclease is able toselectively inactivate by DNA cleavage the gene coding for TCR alpha orTCR beta. According to a preferred embodiment, the rare-cuttingendonuclease is able to selectively inactivate by DNA cleavage the genecoding for TCR alpha. Especially in case of an allogeneic immune cellobtained from a donor, inactivating of at least one gene encoding acomponent of TCR, notably TCR alpha, leads to engineered immune cells,when infused into an allogeneic host, which are non-alloreactive. Thismakes the engineered immune cell particular suitable for allogeneictransplantations, especially because it reduces the risk of graft versushost disease.

A rare-cutting endonuclease to be used in accordance with the presentinvention to inactivate at least one gene encoding a component of theT-cell receptor may, for instance, be a TALE-nuclease, meganuclease,zinc-finger nuclease (ZFN), or RNA guided endonuclease (such as Cas9).

According to a particular embodiment, the rare-cutting endonuclease is aTALE-nuclease.

According to another particular embodiment, the rare-cuttingendonuclease is a homing endonuclease, also known under the name ofmeganuclease.

According to another particular embodiment, the rare-cuttingendonuclease is a zinc-finger nuclease (ZNF).

According to another particular embodiment, the rare-cuttingendonuclease is a RNA guided endonuclease. According to a preferredembodiment, the RNA guided endonuclease is the Cas9/CRISPR complex.

In order to be expressed in the immune cell, such as a T-cell, arare-cutting endonuclease used in accordance with the present inventionto inactive at least one gene encoding a component of the T-cellreceptor may be introduced into the cell by way of an exogenous nucleicacid molecule comprising a nucleotide sequence encoding saidrare-cutting endonuclease. Accordingly, the method of the invention maycomprise introducing into said immune cell an exogenous nucleic acidmolecule comprising a nucleotide sequence encoding a rare-cuttingendonuclease able to selectively inactivate by DNA cleavage, preferablydouble-strand break, at least one gene encoding a component of theT-cell receptor.

As a result, an engineered immune cell, such as a T-cell, is obtainedwhich further expresses a rare-cutting endonuclease able to selectivelyinactivate by DNA cleavage at least one gene encoding a component of theT-cell receptor. In consequence, an engineered immune cell, such as aT-cell, is obtained which is characterized in that at least one geneencoding a component of the T-cell receptor, such as TCR alpha, isinactivated.

It is also contemplated by the present invention that the engineeredimmune cell, such as a T-cell, further expresses a Chimeric AntigenReceptor (CAR) directed against at least one antigen expressed at thesurface of a malignant cell. Hence, in accordance with certainembodiments, the method of the present invention further comprisesintroducing into said immune cell an exogenous nucleic acid moleculecomprising a nucleotide sequence encoding a Chimeric Antigen Receptordirected against at least one antigen expressed at the surface of amalignant cell. According to particular embodiments, said CAR isdirected against an antigen selected from CD19, CD33, CD123, CS1, BCMA,CD38, 5T4, ROR1 and EGFRvIII.

The immune cell to be modified according to the present invention may beany suitable immune cell. For example, the immune cell may be a T-cellor a natural killer (NK) cell. According to certain embodiments, theimmune cell is a T-cell, such as an inflammatory T-lymphocyte, cytotoxicT-lymphocyte, regulatory T-cell or helper T-lymphocyte. According toparticular embodiments, the T-cell is a cytotoxic T-lymphocyte.According to particular embodiments, the T-cell is a CD4+ T-lymphocyte.According to particular embodiments, the T-cell is a CD8+ T-lymphocyte.According to certain other embodiments, the immune cell is a naturalkiller cell.

The immune cell may be extracted from blood. Alternatively, the immunecell may be derived from a stem cell, e.g. by in vitro differentiation.The stem cell can be an adult stem cell, embryonic stem cell, cord bloodstem cell, progenitor cell, bone marrow stem cell, induced pluripotentstem cell, or hematopoietic stem cell. The stem cell may a human ornon-human stem cell. Representative human cells are CD34+ cells.

According to certain embodiments, the immune cell is derived from a stemcell, e.g., by in vitro differentiation. According to particularembodiments, the stem cell is a pluripotent stem cell, such as anembryonic stem cell or induced pluripotent stem cell. According toparticular other embodiments, the stem cell is a multipotent stem cell,such as a haematopoietic stem cell. According to certain otherembodiments, the immune cell is derived from a common lymphoidprogenitor (CLP) cell, e.g., by in vitro differentiation.

According to certain embodiments, the immune cell is a mammalian immunecell. According to particular embodiments, the immune cell is a primateimmune cell. According to more particular embodiments, the immune cellis a human immune cell, such as a human T-cell.

Prior to expansion and genetic modification of the immune cells of theinvention, a source of cells can be obtained from a subject, such as apatient, through a variety of non-limiting methods. An immune cell, suchas a T-cell, can be obtained from a number of non-limiting sources,including peripheral blood mononuclear cells, bone marrow, lymph nodetissue, cord blood, thymus tissue, tissue from a site of infection,ascites, pleural effusion, spleen tissue, and tumors. According tocertain embodiments, any number of immune cell lines available and knownto those skilled in the art, may be used. According to other certainembodiments, the immune cell can be obtained from a healthy donor.According to other certain embodiments, the immune cell can be obtainedfrom a patient diagnosed with malignancy. In other certain embodiments,said cell is part of a mixed population of cells which present differentphenotypic characteristics.

Rare-Cutting Endonuclease

In accordance with certain embodiments of the present invention,rare-cutting endonucleases are employed which are able to selectivelyinactivate by DNA cleavage the gene of interest, such as the geneencoding GCN2.

The term “rare-cutting endonuclease” refers to a wild type or variantenzyme capable of catalyzing the hydrolysis (cleavage) of bonds betweennucleic acids within a DNA or RNA molecule, preferably a DNA molecule.Particularly, said nuclease can be an endonuclease, more preferably arare-cutting endonuclease which is highly specific, recognizing nucleicacid target sites ranging from 10 to 45 base pairs (bp) in length,usually ranging from 10 to 35 base pairs in length, more usually from 12to 20 base pairs. The endonuclease according to the present inventionrecognizes at specific polynucleotide sequences, further referred to as“target sequence” and cleaves nucleic acid inside these target sequencesor into sequences adjacent thereto, depending on the molecular structureof said endonuclease. The rare-cutting endonuclease can recognize andgenerate a single- or double-strand break at specific polynucleotidessequences.

In particular embodiments, a rare-cutting endonuclease according to thepresent invention is a RNA-guided endonuclease such as the Cas9/CRISPRcomplex. RNA guided endonucleases constitute a new generation of genomeengineering tool where an endonuclease associates with a RNA molecule.In this system, the RNA molecule nucleotide sequence determines thetarget specificity and activates the endonuclease (Gasiunas, Barrangouet al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et al. 2013; Mali,Yang et al. 2013). Cas9, also named Csn1 is a large protein thatparticipates in both crRNA biogenesis and in the destruction of invadingDNA. Cas9 has been described in different bacterial species such as S.thermophiles, Listeria innocua (Gasiunas, Barrangou et al. 2012; Jinek,Chylinski et al. 2012) and S. Pyogenes (Deltcheva, Chylinski et al.2011). The large Cas9 protein (>1200 amino acids) contains two predictednuclease domains, namely HNH (McrA-like) nuclease domain that is locatedin the middle of the protein and a splitted RuvC-like nuclease domain(RNase H fold). Cas9 variant can be a Cas9 endonuclease that does notnaturally exist in nature and that is obtained by protein engineering orby random mutagenesis. Cas9 variants according to the invention can forexample be obtained by mutations i.e. deletions from, or insertions orsubstitutions of at least one residue in the amino acid sequence of a S.pyogenes Cas9 endonuclease (COG3513).

In other particular embodiments, a rare-cutting endonuclease can also bea homing endonuclease, also known under the name of meganuclease. Suchhoming endonucleases are well-known to the art (Stoddard, B. L., 2005).Homing endonucleases are highly specific, recognizing DNA target sitesranging from 12 to 45 base pairs (bp) in length, usually ranging from 14to 40 bp in length. The homing endonuclease according to the inventionmay for example correspond to a LAGLIDADG endonuclease, to a HNHendonuclease, or to a GIY-YIG endonuclease. Preferred homingendonuclease according to the present invention can be an I-CreIvariant. A “variant” endonuclease, i.e. an endonuclease that does notnaturally exist in nature and that is obtained by genetic engineering orby random mutagenesis can bind DNA sequences different from thatrecognized by wild-type endonucleases (see international applicationWO2006/097854).

In other particular embodiments, a rare-cutting endonuclease can be a“Zinc Finger Nuclease” (ZFN). ZNFs are generally a fusion between thecleavage domain of the type IIS restriction enzyme, Fokl, and a DNArecognition domain containing 3 or more C2H2 zinc finger motifs. Theheterodimerization at a particular position in the DNA of two individualZFNs in precise orientation and spacing leads to a double-strand break(DSB) in the DNA. The use of such chimeric endonucleases have beenextensively reported in the art as reviewed by Urnov et al. (2010).Standard ZFNs fuse the cleavage domain to the C-terminus of each zincfinger domain. In order to allow the two cleavage domains to dimerizeand cleave DNA, the two individual ZFNs bind opposite strands of DNAwith their C-termini a certain distance apart. The most commonly usedlinker sequences between the zinc finger domain and the cleavage domainrequires the 5′ edge of each binding site to be separated by 5 to 7 bp.The most straightforward method to generate new zinc-finger arrays is tocombine smaller zinc-finger “modules” of known specificity. The mostcommon modular assembly process involves combining three separate zincfingers that can each recognize a 3 base pair DNA sequence to generate a3-finger array that can recognize a 9 base pair target site. Numerousselection methods have been used to generate zinc-finger arrays capableof targeting desired sequences. Initial selection efforts utilized phagedisplay to select proteins that bound a given DNA target from a largepool of partially randomized zinc-finger arrays. More recent effortshave utilized yeast one-hybrid systems, bacterial one-hybrid andtwo-hybrid systems, and mammalian cells.

In other particular embodiments, a rare-cutting endonuclease is a“TALE-nuclease” (see, e.g., WO2011159369) or a “MBBBD-nuclease” (see,e.g., WO2014018601) resulting from the fusion of a DNA binding domaintypically derived from Transcription Activator Like Effector proteins(TALE) or from a Modular Base-per-Base Binding domain (MBBBD), with acatalytic domain having endonuclease activity. Such catalytic domainusually comes from enzymes, such as for instance I-TevI, ColE7, NucA andFok-I. TALE-nuclease can be formed under monomeric or dimeric formsdepending of the selected catalytic domain (WO2012138927). Suchengineered TALE-nucleases are commercially available under the tradename TALEN™ (Cellectis, 8 rue de la Croix Jarry, 75013 Paris, France).In general, the DNA binding domain is derived from a TranscriptionActivator like Effector (TALE), wherein sequence specificity is drivenby a series of 33-35 amino acids repeats originating from Xanthomonas orRalstonia bacterial proteins AvrBs3, PthXo1, AvrHah1, PthA, Tal1c asnon-limiting examples. These repeats differ essentially by two aminoacids positions that specify an interaction with a base pair (Boch,Scholze et al. 2009; Moscou and Bogdanove 2009). Each base pair in theDNA target is contacted by a single repeat, with the specificityresulting from the two variant amino acids of the repeat (the so-calledrepeat variable dipeptide, RVD). TALE binding domains may furthercomprise an N-terminal translocation domain responsible for therequirement of a first thymine base (T0) of the targeted sequence and aC-terminal domain that containing a nuclear localization signals (NLS).A TALE nucleic acid binding domain generally corresponds to anengineered core TALE scaffold comprising a plurality of TALE repeatsequences, each repeat comprising a RVD specific to each nucleotidesbase of a TALE recognition site. In the present invention, each TALErepeat sequence of said core scaffold is made of 30 to 42 amino acids,more preferably 33 or 34 wherein two critical amino acids (the so-calledrepeat variable dipeptide, RVD) located at positions 12 and 13 mediatesthe recognition of one nucleotide of said TALE binding site sequence;equivalent two critical amino acids can be located at positions otherthan 12 and 13 specially in TALE repeat sequence taller than 33 or 34amino acids long. Preferably, RVDs associated with recognition of thedifferent nucleotides are HD for recognizing C, NG for recognizing T, NIfor recognizing A, NN for recognizing G or A. In another embodiment,critical amino acids 12 and 13 can be mutated towards other amino acidresidues in order to modulate their specificity towards nucleotides A,T, C and G and in particular to enhance this specificity. A TALE nucleicacid binding domain usually comprises between 8 and 30 TALE repeatsequences. More preferably, said core scaffold of the present inventioncomprises between 8 and 20 TALE repeat sequences; again more preferably15 TALE repeat sequences. It can also comprise an additional singletruncated TALE repeat sequence made of 20 amino acids located at theC-terminus of said set of TALE repeat sequences, i.e. an additionalC-terminal half-TALE repeat sequence. Other modular base-per-basespecific nucleic acid binding domains (MBBBD) are described in WO2014018601. Said MBBBD can be engineered, for instance, from newlyidentified proteins, namely EAV36_BURRH, E5AW43_BURRH, E5AW45_BURRH andE5AW46_BURRH proteins from the recently sequenced genome of theendosynnbiont fungi Burkholderia Rhizoxinica. These nucleic acid bindingpolypeptides comprise modules of about 31 to 33 amino acids that arebase specific. These modules display less than 40% sequence identitywith Xanthomonas TALE common repeats and present more polypeptidessequence variability. The different domains from the above proteins(modules, N and C-terminals) from Burkholderia and Xanthomonas areuseful to engineer new proteins or scaffolds having binding propertiesto specific nucleic acid sequences and may be combined to form chimericTALE-MBBBD proteins.

As far as TALE-nucleases are concerned, suitable target sequences in thegene of interest may be identified by available software tools. Forexample, the software tool “Target Finder”, which is provide as part ofthe TAL Effector Nucleotide Targeter (TALENT) 2.0 software packagedeveloped by Doyle et al. (2012), is a web-based tool (accessiblethought, e.g., https://tale-nt.cac.cornell.edu/) which allows theidentification of target sequences of TALE nucleases. Custom madeTALE-nucleases may be ordered from Cellectis Bioresearch, 8 rue de laCroix Jarry, 75013 Paris, France.

Exemplary, non-limiting target sequences within the human GCN2 gene forinactivation by a rare-cutting endonuclease are set forth in SEQ ID NO:3 and SEQ ID NO: 4.

According to particular embodiments, the rare-cutting endonucleasetargets (e.g., binds to) a sequence set forth in SEQ ID NO: 3. Accordingto other particular embodiments, the rare-cutting endonuclease targets(e.g., binds to) a sequence set forth in SEQ ID NO: 4.

Chimeric Antigen Receptors (CARs)

Adoptive immunotherapy, which involves the transfer of autologousantigen-specific T-cells generated ex vivo, is a promising strategy totreat cancer or viral infections. The T-cells used for adoptiveimmunotherapy can be generated either by expansion of antigen-specific Tcells or redirection of T cells through genetic engineering (Park,Rosenberg et al. 2011). Transfer of viral antigen specific T-cells is awell-established procedure used for the treatment of transplantassociated viral infections and rare viral-related malignancies.Similarly, isolation and transfer of tumor specific T cells has beenshown to be successful in treating melanoma.

Novel specificities in T-cells have been successfully generated throughthe genetic transfer of transgenic T-cell receptors or chimeric antigenreceptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptorsconsisting of a targeting moiety that is associated with one or moresignaling domains in a single fusion molecule. In general, the bindingmoiety of a CAR consists of an antigen-binding domain of a single-chainantibody (scFv), comprising the light and variable fragments of amonoclonal antibody joined by a flexible linker. Binding moieties basedon receptor or ligand domains have also been used successfully. Thesignaling domains for first generation CARs are derived from thecytoplasmic region of the CD3zeta or the Fc receptor gamma chains. Firstgeneration CARs have been shown to successfully redirect T cellcytotoxicity, however, they failed to provide prolonged expansion andanti-tumor activity in vivo. Signaling domains from co-stimulatorymolecules including CD28, OX-40 (CD134), and 4-1BB (CD137) have beenadded alone (second generation) or in combination (third generation) toenhance survival and increase proliferation of CAR modified T-cells.CARs have successfully allowed T-cells to be redirected against antigensexpressed at the surface of tumor cells from various malignanciesincluding lymphomas and solid tumors (Jena, Dotti et al. 2010).

According to certain embodiments, the Chimeric Antigen Receptorexpressed by the engineered immune cell is directed against an antigenselected from CD19, CD33, CD123, CS1, BCMA, CD38, 5T4, ROR1 andEGFRvIII. According to particular embodiments, the Chimeric AntigenReceptor expressed by the engineered immune cell is directed againstCD33. According to other particular embodiments, the Chimeric AntigenReceptor expressed by the engineered immune cell is directed againstCS1. According to other particular embodiments, the Chimeric AntigenReceptor expressed by the engineered immune cell is directed againstBCMA. According to other particular embodiments, the Chimeric AntigenReceptor expressed by the engineered immune cell is directed againstCD38.

According to certain embodiments, the Chimeric Antigen Receptorexpressed by the engineered immune cell is directed against an antigencommonly expressed at the surface of solid tumor cells, such as 5T4,ROR1 and EGFRvIII. According to particular embodiments, the ChimericAntigen Receptor expressed by the engineered immune cell is directedagainst 5T4. According to other particular embodiments, the ChimericAntigen Receptor expressed by the engineered immune cell is directedagainst ROR1. According to other particular embodiments, the ChimericAntigen Receptor expressed by the engineered immune cell is directedagainst EGFRvIII.

According to certain other embodiments, the Chimeric Antigen Receptorexpressed by the engineered immune cell is directed against an antigencommonly expressed at the surface of liquid tumors, such as CD123.According to particular embodiments, the Chimeric Antigen Receptorexpressed by the engineered immune cell is directed against CD123.

CD19 is an attractive target for immunotherapy because the vast majorityof B-acute lymphoblastic leukemia (B-ALL) uniformly express CD19,whereas expression is absent on non hematopoietic cells, as well asmyeloid, erythroid, and T cells, and bone marrow stem cells. Clinicaltrials targeting CD19 on B-cell malignancies are underway withencouraging anti-tumor responses. T-cells genetically modified toexpress a chimeric antigen receptor (CAR) with specificity derived fromthe scFv region of a CD19-specific mouse monoclonal antibody FMC63 aredescribed in WO2013/126712.

Therefore, in accordance with particular embodiments, the ChimericAntigen Receptor expressed by the engineered immune cell is directedagainst the B-lymphocyte antigen CD19.

In accordance with certain embodiments, the Chimeric Antigen Receptor isa single chain Chimeric Antigen Receptor. As an example of single-chainChimeric Antigen Receptor to be expressed in the engineered immune cellaccording to the present invention is a single polypeptide thatcomprises at least one extracellular ligand binding domain, atransmembrane domain and at least one signal transducing domain, whereinsaid extracellular ligand binding domain comprises a scFV derived fromthe specific anti-CD19 monoclonal antibody 4G7. Once transduced into theimmune cell, for instance by using retroviral or lentiviraltransduction, this CAR contributes to the recognition of CD19 antigenpresent at the surface of malignant B-cells involved in lymphoma orleukemia.

In accordance with particular embodiments, the Chimeric Antigen Receptoris a polypeptide comprising the amino acid sequence forth in SEQ ID NO:5 or a variant thereof comprising an amino acid sequence that has atleast 70%, such as at least 80%, at least 90%, at least 95%, or at least99%, sequence identity with the amino acid sequence set forth in SEQ IDNO: 5 over the entire length of SEQ ID NO: 5. Preferably, the variant iscapable of binding CD19.

A particularly preferred Chimeric Antigen Receptor is a polypeptidecomprising the amino acid sequence set forth in SEQ ID NO: 6 or avariant thereof comprising an amino acid sequence that has at least 80%,such as at least 90%, at least 95%, or at least 99%, sequence identitywith the amino acid sequence set forth in SEQ ID NO: 6 over the entirelength of SEQ ID NO: 6. Such variant may differ from the polypeptide setforth in SEQ ID NO: 6 in the substitution of at least one, at least twoor at least three amino acid residue(s). Preferably, said variant iscapable of binding CD19.

In accordance with other certain embodiments, the Chimeric AntigenReceptor may be directed against another antigen expressed at thesurface of a malignant or infected cell, such as a cluster ofdifferentiation molecule, such as CD16, CD64, CD78, CD96, CLL1, CD116,CD117, CD71, CD45, CD71, CD123 and CD138, a tumor-associated surfaceantigen, such as ErbB2 (HER2/neu), carcinoembryonic antigen (CEA),epithelial cell adhesion molecule (EpCAM), epidermal growth factorreceptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40,disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72,glycosphingolipids, glioma-associated antigen, β-human chorionicgonadotropin, alphafetoprotein (AFP), lectin-reactive AFP,thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase,RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF,prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53,prostein, PSMA, surviving and telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22,insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, amajor histocompatibility complex (MHC) molecule presenting atumor-specific peptide epitope, 5T4, ROR1, Nkp30, NKG2D, tumor stromalantigens, the extra domain A (EDA) and extra domain B (EDB) offibronectin and the A1 domain of tenascin-C(TnC A1) and fibroblastassociated protein (fap); a lineage-specific or tissue specific antigensuch as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4,B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, a majorhistocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF 17),multiple myeloma or lymphoblastic leukaemia antigen, such as oneselected from TNFRSF17 (UNIPROT Q02223), SLAMF7 (UNIPROT Q9NQ25), GPRC5D(UNIPROT Q9NZD1), FKBP11 (UNIPROT Q9NYL4), KAMP3, ITGA8 (UNIPROTP53708), and FCRL5 (UNIPROT Q68SN8). a virus-specific surface antigensuch as an HIV-specific antigen (such as HIV gp120); an EBV-specificantigen, a CMV-specific antigen, a HPV-specific antigen, a LasseVirus-specific antigen, an Influenza Virus-specific antigen as well asany derivate or variant of these surface antigens.

In other certain embodiments, the Chimeric Antigen Receptor is amulti-chain Chimeric Antigen Receptor. Chimeric Antigen Receptors fromthe prior art introduced in T-cells have been formed of single chainpolypeptides that necessitate serial appending of signaling domains.However, by moving signaling domains from their natural juxtamembraneposition may interfere with their function. To overcome this drawback,the applicant recently designed a multi-chain CAR derived from FcεRI toallow normal juxtamembrane position of all relevant signaling domains.In this new architecture, the high affinity IgE binding domain of FcεRIalpha chain is replaced by an extracellular ligand-binding domain suchas scFv to redirect T-cell specificity against cell targets and the Nand/or C-termini tails of FcεRI beta chain are used to placecostimulatory signals in normal juxtamembrane positions as described inWO 2013/176916.

Accordingly, a CAR expressed by the engineered immune cell according tothe invention can be a multi-chain chimeric antigen receptorparticularly adapted to the production and expansion of engineeredimmune cells of the present invention. Such multi-chain CARs comprise atleast two of the following components:

-   -   a) one polypeptide comprising the transmembrembrane domain of        FcεRI alpha chain and an extracellular ligand-binding domain,    -   b) one polypeptide comprising a part of N- and C-terminal        cytoplasmic tail and the transmembrane domain of FcεRI beta        chain and/or    -   c) at least two polypeptides comprising each a part of        intracytoplasmic tail and the transmembrane domain of FcεRI        gamma chain, whereby different polypeptides multimerize together        spontaneously to form dimeric, trimeric or tetrameric CAR.

According to such architectures, ligands binding domains and signalingdomains are born on separate polypeptides. The different polypeptidesare anchored into the membrane in a close proximity allowinginteractions with each other. In such architectures, the signaling andco-stimulatory domains can be in juxtamembrane positions (i.e. adjacentto the cell membrane on the internal side of it), which is deemed toallow improved function of co-stimulatory domains. The multi-subunitarchitecture also offers more flexibility and possibilities of designingCARs with more control on T-cell activation. For instance, it ispossible to include several extracellular antigen recognition domainshaving different specificity to obtain a multi-specific CARarchitecture. It is also possible to control the relative ratio betweenthe different subunits into the multi-chain CAR. This type ofarchitecture is more detailed in WO2014039523.

The assembly of the different chains as part of a single multi-chain CARis made possible, for instance, by using the different alpha, beta andgamma chains of the high affinity receptor for IgE (FcεRI) (Metzger,Alcaraz et al. 1986) to which are fused the signaling and co-stimulatorydomains. The gamma chain comprises a transmembrane region andcytoplasmic tail containing one immunoreceptor tyrosine-based activationmotif (ITAM) (Cambier 1995).

The multi-chain CAR can comprise several extracellular ligand-bindingdomains, to simultaneously bind different elements in target therebyaugmenting immune cell activation and function. In one embodiment, theextracellular ligand-binding domains can be placed in tandem on the sametransmembrane polypeptide, and optionally can be separated by a linker.In another embodiment, said different extracellular ligand-bindingdomains can be placed on different transmembrane polypeptides composingthe multi-chain CAR.

The signal transducing domain or intracellular signaling domain of themulti-chain CAR(s) of the invention is responsible for intracellularsignaling following the binding of extracellular ligand binding domainto the target resulting in the activation of the immune cell and immuneresponse. In other words, the signal transducing domain is responsiblefor the activation of at least one of the normal effector functions ofthe immune cell in which the multi-chain CAR is expressed. For example,the effector function of a T cell can be a cytolytic activity or helperactivity including the secretion of cytokines.

In the present application, the term “signal transducing domain” refersto the portion of a protein which transduces the effector signalfunction signal and directs the cell to perform a specialized function.

Preferred examples of signal transducing domain for use in single ormulti-chain CAR can be the cytoplasmic sequences of the Fc receptor or Tcell receptor and co-receptors that act in concert to initiate signaltransduction following antigen receptor engagement, as well as anyderivate or variant of these sequences and any synthetic sequence thatas the same functional capability. Signal transduction domain comprisestwo distinct classes of cytoplasmic signaling sequence, those thatinitiate antigen-dependent primary activation, and those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal. Primary cytoplasmic signaling sequence can comprise signalingmotifs which are known as immunoreceptor tyrosine-based activationmotifs of ITAMs. ITAMs are well defined signaling motifs found in theintracytoplasmic tail of a variety of receptors that serve as bindingsites for syk/zap70 class tyrosine kinases. Examples of ITAM used in theinvention can include as non-limiting examples those derived fromTCRzeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon,CD5, CD22, CD79a, CD79b and CD66d. According to particular embodiments,the signaling transducing domain of the multi-chain CAR can comprise theCD3zeta signaling domain, or the intracytoplasmic domain of the FcεRIbeta or gamma chains.

According to particular embodiments, the signal transduction domain ofmulti-chain CARs of the present invention comprises a co-stimulatorysignal molecule. A co-stimulatory molecule is a cell surface moleculeother than an antigen receptor or their ligands that is required for anefficient immune response.

Ligand binding-domains can be any antigen receptor previously used, andreferred to, with respect to single-chain CAR referred to in theliterature, in particular scFv from monoclonal antibodies.

Delivery Methods

The inventors have considered any means known in the art to allowdelivery inside cells or subcellular compartments of said cells thenucleic acid molecules employed in accordance with the invention. Thesemeans include viral transduction, electroporation and also liposomaldelivery means, polymeric carriers, chemical carriers, lipoplexes,polyplexes, dendrimers, nanoparticles, emulsion, natural endocytosis orphagocytose pathway as non-limiting examples.

In accordance with the present invention, the nucleic acid moleculesdetailed herein may be introduced in the immune cell by any suitablemethods known in the art. Suitable, non-limiting methods for introducinga nucleic acid molecule into an immune cell include stabletransformation methods, wherein the nucleic acid molecule is integratedinto the genome of the cell, transient transformation methods whereinthe nucleic acid molecule is not integrated into the genome of the celland virus mediated methods. Said nucleic acid molecule may be introducedinto a cell by, for example, a recombinant viral vector (e.g.,retroviruses, adenoviruses), liposome and the like. Transienttransformation methods include, for example, microinjection,electroporation or particle bombardment. In certain embodiments, thenucleic acid molecule is a vector, such as a viral vector or plasmid.Suitably, said vector is an expression vector enabling the expression ofthe respective polypeptide(s) or protein(s) detailed herein by theimmune cell.

A nucleic acid molecule introduced into the immune cell may be DNA orRNA. In certain embodiments, a nucleic acid molecule introduced into theimmune cell is DNA. In certain other embodiments, a nucleic acidmolecule introduced into the immune cell is RNA, and in particular anmRNA encoding a polypeptide or protein detailed herein, which mRNA isintroduced directly into the immune cell, for example byelectroporation. A suitable electroporation technique is described, forexample, in International Publication WO2013/176915 (in particular thesection titled “Electroporation” bridging pages 29 to 30). A particularnucleic acid molecule which may be an mRNA is the nucleic acid moleculecomprising a nucleotide sequence encoding a rare-cutting endonucleaseable to selectively inactivate by DNA cleavage the gene encoding GCN2.Another particular nucleic acid molecule which may be an mRNA is thenucleic acid molecule comprising a nucleotide sequence encoding arare-cutting endonuclease able to selectively inactivate by DNA cleavagethe gene encoding PRDM1. A yet other particular nucleic acid moleculewhich may be an mRNA is the nucleic acid molecule comprising anucleotide sequence encoding a rare-cutting endonuclease able toselectively inactivate by DNA cleavage at least one gene coding for onecomponent of the T-cell receptor.

Nucleic acid molecules encoding the endonucleases of the presentinvention may be transfected under mRNA form in order to obtaintransient expression and avoid chromosomal integration of foreign DNA,for example by electroporation. In this respect, the cytoPulsetechnology may be used which allows, by the use of pulsed electricfields, to transiently permeabilize living cells for delivery ofmaterial into the cells (U.S. Pat. No. 6,010,613 and WO 2004/083379).

Non Alloreactive Immune Cells:

T-cell receptors are cell surface receptors that participate in theactivation of T-cells in response to the presentation of antigen. TheTCR is generally made from two chains, alpha and beta, which assemble toform a heterodimer and associates with the CD3-transducing subunits toform the T-cell receptor complex present on the cell surface. Each alphaand beta chain of the TCR consists of an immunoglobulin-like N-terminalvariable (V) and constant (C) region, a hydrophobic transmembranedomain, and a short cytoplasmic region. As for immunoglobulin molecules,the variable region of the alpha and beta chains are generated by V(D)Jrecombination, creating a large diversity of antigen specificitieswithin the population of T cells. However, in contrast toimmunoglobulins that recognize intact antigen, T cells are activated byprocessed peptide fragments in association with an MHC molecule,introducing an extra dimension to antigen recognition by T cells, knownas MHC restriction. Recognition of MHC disparities between the donor andrecipient through the T cell receptor leads to T cell proliferation andthe potential development of GVHD. It has been shown that normal surfaceexpression of the TCR depends on the coordinated synthesis and assemblyof all seven components of the complex (Ashwell and Klusner 1990). Theinactivation of TCR alpha or TCR beta can result in the elimination ofthe TCR from the surface of T cells preventing recognition ofalloantigen and thus GVHD.

Thus, still according to the invention, engraftment of an immune cell,in particular a T-cells, may be improved by inactivating at least onegene encoding a TCR component. TCR is rendered not functional in thecells by inactivating the gene encoding TCR alpha or TCR beta.

With respect to the use of Cas9/CRISPR system, applicant has determinedappropriate target sequences within the 3 exons encoding TCR, allowing asignificant reduction of toxicity in living cells, while retainingcleavage efficiency. The preferred target sequences are noted in Table 1(+ for lower ratio of TCR negative cells, ++ for intermediate ratio, +++for higher ratio).

TABLE 1 appropriate target sequences for the guide RNA using Cas9 in T-cellsExon TCR Position Strand Target genomic sequence SEQ ID efficiency Ex178 −1 GAGAATCAAAATCGGTGAATAGG 7 +++ Ex3 26 1 TTCAAAACCTGTCAGTGATTGGG 8+++ Ex1 153 1 TGTGCTAGACATGAGGTCTATGG 9 +++ Ex3 74 −1CGTCATGAGCAGATTAAACCCGG 10 +++ Ex1 4 −1 TCAGGGTTCTGGATATCTGTGGG 11 +++Ex1 5 −1 GTCAGGGTTCTGGATATCTGTGG 12 +++ Ex3 33 −1TTCGGAACCCAATCACTGACAGG 13 +++ Ex3 60 −1 TAAACCCGGCCACTTTCAGGAGG 14 +++Ex1 200 −1 AAAGTCAGATTTGTTGCTCCAGG 15 ++ Ex1 102 1AACAAATGTGTCACAAAGTAAGG 16 ++ Ex1 39 −1 TGGATTTAGAGTCTCTCAGCTGG 17 ++Ex1 59 −1 TAGGCAGACAGACTTGTCACTGG 18 ++ Ex1 22 −1AGCTGGTACACGGCAGGGTCAGG 19 ++ Ex1 21 −1 GCTGGTACACGGCAGGGTCAGGG 20 ++Ex1 28 −1 TCTCTCAGCTGGTACACGGCAGG 21 ++ Ex3 25 1 TTTCAAAACCTGTCAGTGATTGG22 ++ Ex3 63 −1 GATTAAACCCGGCCACTTTCAGG 23 ++ Ex2 17 −1CTCGACCAGCTTGACATCACAGG 24 ++ Ex1 32 −1 AGAGTCTCTCAGCTGGTACACGG 25 ++Ex1 27 −1 CTCTCAGCTGGTACACGGCAGGG 26 ++ Ex2 12 1 AAGTTCCTGTGATGTCAAGCTGG27 ++ Ex3 55 1 ATCCTCCTCCTGAAAGTGGCCGG 28 ++ Ex3 86 1TGCTCATGACGCTGCGGCTGTGG 29 ++ Ex1 146 1 ACAAAACTGTGCTAGACATGAGG 30 + Ex186 −1 ATTTGTTTGAGAATCAAAATCGG 31 + Ex2 3 −1 CATCACAGGAACTTTCTAAAAGG 32 +Ex2 34 1 GTCGAGAAAAGCTTTGAAACAGG 33 + Ex3 51 −1 CCACTTTCAGGAGGAGGATTCGG34 + Ex3 18 −1 CTGACAGGTTTTGAAAGTTTAGG 35 + Ex2 43 1AGCTTTGAAACAGGTAAGACAGG 36 + Ex1 236 −1 TGGAATAATGCTGTTGTTGAAGG 37 + Ex1182 1 AGAGCAACAGTGCTGTGGCCTGG 38 + Ex3 103 1 CTGTGGTCCAGCTGAGGTGAGGG39 + Ex3 97 1 CTGCGGCTGTGGTCCAGCTGAGG 40 + Ex3 104 1TGTGGTCCAGCTGAGGTGAGGGG 41 + Ex1 267 1 CTTCTTCCCCAGCCCAGGTAAGG 42 + Exl15 −1 ACACGGCAGGGTCAGGGTTCTGG 43 + Ex1 177 1 CTTCAAGAGCAACAGTGCTGTGG44 + Ex1 256 −1 CTGGGGAAGAAGGTGTCTTCTGG 45 + Ex3 56 1TCCTCCTCCTGAAAGTGGCCGGG 46 + Ex3 80 1 TTAATCTGCTCATGACGCTGCGG 47 + Ex357 −1 ACCCGGCCACTTTCAGGAGGAGG 48 + Ex1 268 1 TTCTTCCCCAGCCCAGGTAAGGG49 + Ex1 266 −1 CTTACCTGGGCTGGGGAAGAAGG 50 + Ex1 262 1GACACCTTCTTCCCCAGCCCAGG 51 + Ex3 102 1 GCTGTGGTCCAGCTGAGGTGAGG 52 + Ex351 1 CCGAATCCTCCTCCTGAAAGTGG 53 +

MHC antigens are also proteins that played a major role intransplantation reactions. Rejection is mediated by T cells reacting tothe histocompatibility antigens on the surface of implanted tissues, andthe largest group of these antigens is the major histocompatibilityantigens (MHC). These proteins are expressed on the surface of allhigher vertebrates and are called HLA antigens (for human leukocyteantigens) in human cells. Like TCR, the MHC proteins serve a vital rolein T cell stimulation. Antigen presenting cells (often dendritic cells)display peptides that are the degradation products of foreign proteinson the cell surface on the MHC. In the presence of a co-stimulatorysignal, the T cell becomes activated, and will act on a target cell thatalso displays that same peptide/MHC complex. For example, a stimulated Thelper cell will target a macrophage displaying an antigen inconjunction with its MHC, or a cytotoxic T cell (CTL) will act on avirally infected cell displaying foreign viral peptides.

Thus, in order to provide less alloreactive T-cells, the method of theinvention can further comprise the step of inactivating or mutating atleast one HLA gene.

The class I HLA gene cluster in humans comprises three major loci, B, Cand A, as well as several minor loci. The class II HLA cluster alsocomprises three major loci, DP, DQ and DR, and both the class I andclass II gene clusters are polymorphic, in that there are severaldifferent alleles of both the class I and II genes within thepopulation. There are also several accessory proteins that play a rolein HLA functioning as well. The TapI and Tap2 subunits are parts of theTAP transporter complex that is essential in loading peptide antigens onto the class I HLA complexes, and the LMP2 and LMP7 proteosome subunitsplay roles in the proteolytic degradation of antigens into peptides fordisplay on the HLA. Reduction in LMP7 has been shown to reduce theamount of MHC class I at the cell surface, perhaps through a lack ofstabilization (Fehling et al. (1999) Science 265:1234-1237). In additionto TAP and LMP, there is the tapasin gene, whose product forms a bridgebetween the TAP complex and the HLA class I chains and enhances peptideloading. Reduction in tapasin results in cells with impaired MHC class Iassembly, reduced cell surface expression of the MHC class I andimpaired immune responses (Grandea et al. (2000) Immunity 13:213-222 andGarbi et al. (2000) Nat. Immunol. 1:234-238). Any of the above genes maybe inactivated as part of the present invention as disclosed, forinstance in WO 2012/012667.

Activation and Expansion of Immune Cells

The method according to the invention may include a further step ofactivating and/or expanding the immune cell(s). This can be done priorto or after genetic modification of the immune cell(s), using themethods as described, for example, in U.S. Pat. Nos. 6,352,694;6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681;7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223;6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application PublicationNo. 20060121005. According to these methods, the immune cells of theinvention can be expanded by contact with a surface having attachedthereto an agent that stimulates a CD3 TCR complex associated signal anda ligand that stimulates a co-stimulatory molecule on the surface of theimmune cells.

In particular, T-cell populations may be stimulated in vitro such as bycontact with an anti-CD3 antibody, or antigen-binding fragment thereof,or an anti-CD2 antibody immobilized on a surface, or by contact with aprotein kinase C activator (e.g., bryostatin) in conjunction with acalcium ionophore. For co-stimulation of an accessory molecule on thesurface of the T-cells cells, a ligand that binds the accessory moleculeis used. For example, a population of T cells can be contacted with ananti-CD3 antibody and an anti-CD28 antibody, under conditionsappropriate for stimulating proliferation of the T cells. To stimulateproliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3antibody and an anti-CD28 antibody. For example, the agents providingeach signal may be in solution or coupled to a surface. As those ofordinary skill in the art can readily appreciate, the ratio of particlesto cells may depend on particle size relative to the target cell. Infurther embodiments of the present invention, the cells, such as Tcells, are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. Cell surface proteinsmay be ligated by allowing paramagnetic beads to which anti-CD3 andanti-CD28 are attached (3×28 beads) to contact the T cells. In oneembodiment the cells (for example, 4 to 10 T cells) and beads (forexample, DYNABEADS® M-450 CD3/CD28 T paramagnetic beads at a ratio of1:1) are combined in a buffer, preferably PBS (without divalent cationssuch as, calcium and magnesium). Again, those of ordinary skill in theart can readily appreciate any cell concentration may be used. Themixture may be cultured for several hours (about 3 hours) to about 14days or any hourly integer value in between. In another embodiment, themixture may be cultured for 21 days. Conditions appropriate for T cellculture include an appropriate media (e.g., Minimal Essential Media orRPMI Media 1640 or, X-vivo 5, (Lonza)) that may contain factorsnecessary for proliferation and viability, including serum (e.g., fetalbovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, 1L-4,1L-7, GM-CSF, -10, -2, 1L-15, TGFp, and TNF- or any other additives forthe growth of cells known to the skilled artisan. Other additives forthe growth of cells include, but are not limited to, surfactant,plasmanate, and reducing agents such as N-acetyl-cysteine and2-mercaptoethanoi. Media can include RPMI 1640, A1M-V, DMEM, MEM, a-MEM,F-12, X-Vivo 1, and X-Vivo 20, Optimizer, with added amino acids, sodiumpyruvate, and vitamins, either serum-free or supplemented with anappropriate amount of serum (or plasma) or a defined set of hormones,and/or an amount of cytokine(s) sufficient for the growth and expansionof T cells. Antibiotics, e.g., penicillin and streptomycin, are includedonly in experimental cultures, not in cultures of cells that are to beinfused into a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2). T cells that havebeen exposed to varied stimulation times may exhibit differentcharacteristics

In another particular embodiment, said immune cell(s) can be expanded byco-culturing with tissue or cells. Said cells can also be expanded invivo, for example in the subject's blood after administrating said cellinto the subject.

Engineered Immune Cells

As a result of the present invention, engineered immune cells can beobtained having improved characteristics. In particular, the presentinvention provides an engineered, preferably isolated, immune cell whichis characterized in that a gene encoding GCN2 and/or a gene encodingPRDM1 is inactivated or repressed.

According to certain embodiments, an engineered immune cell is providedwherein a gene encoding GCN2 (e.g., the human GCN2 gene) is inactivated.

According to certain other embodiments, an engineered immune cell isprovided wherein a gene encoding GCN2 (e.g., the human GCN2 gene) isrepressed.

According to certain other embodiments, an engineered immune cell isprovided wherein a gene encoding PRDM1 (e.g., the human PRDM1 gene) isinactivated.

According to certain other embodiments, an engineered immune cell isprovided wherein a gene encoding PRDM1 (e.g., the human PRDM1 gene) isrepressed.

According to certain other embodiments, an engineered immune cell isprovided wherein both a gene encoding GCN2 (e.g., the human GCN2 gene)and a gene encoding PRDM1 (e.g., the human PRDM1 gene) are inactivated.

According to certain other embodiments, an engineered immune cell isprovided wherein both a gene encoding GCN2 (e.g., the human GCN2 gene)and a gene encoding PRDM1 (e.g., the human PRDM1 gene) are repressed.

According to certain other embodiments, an engineered immune cell isprovided wherein a gene encoding GCN2 (e.g., the human GCN2 gene) isinactivated and a gene encoding PRDM1 (e.g., the human PRDM1 gene) isrepressed.

According to certain other embodiments, an engineered immune cell isprovided wherein a gene encoding GCN2 (e.g., the human GCN2 gene) isrepressed and a gene encoding PRDM1 (e.g., the human PRDM1 gene) isinactivated.

According to certain embodiments, an engineered immune cell is obtainedwhich expresses a rare-cutting endonuclease able to selectivelyinactivate by DNA cleavage, preferably double-strand break, a geneencoding GCN2. According to particular embodiments, said immune cellcomprises an exogenous nucleic acid molecule comprising a nucleotidesequence encoding said rare-cutting endonuclease. According to moreparticular embodiments, said rare-cutting endonuclease is aTALE-nuclease, meganuclease, zinc-finger nuclease (ZFN), or RNA guidedendonuclease. Hence, in accordance with a specific embodiment, therare-cutting endonuclease is a TALE-nuclease. In accordance with anotherspecific embodiment, the rare-cutting endonuclease is a meganuclease. Inaccordance with another specific embodiment, the rare-cuttingendonuclease is a zinc-finger nuclease. In accordance with yet anotherspecific embodiment, the rare-cutting endonuclease is a RNA guidedendonuclease, such as Cas9.

According to certain embodiments, an engineered immune cell is obtainedwhich expresses a rare-cutting endonuclease able to selectivelyinactivate by DNA cleavage, preferably double-strand break, a geneencoding PRDM1. According to particular embodiments, said immune cellcomprises an exogenous nucleic acid molecule comprising a nucleotidesequence encoding said rare-cutting endonuclease. According to moreparticular embodiments, said rare-cutting endonuclease is aTALE-nuclease, meganuclease, zinc-finger nuclease (ZFN), or RNA guidedendonuclease. Hence, in accordance with a specific embodiment, therare-cutting endonuclease is a TALE-nuclease. In accordance with anotherspecific embodiment, the rare-cutting endonuclease is a meganuclease. Inaccordance with another specific embodiment, the rare-cuttingendonuclease is a zinc-finger nuclease. In accordance with yet anotherspecific embodiment, the rare-cutting endonuclease is a RNA guidedendonuclease, such as Cas9.

According to certain other embodiments, an engineered immune cell isobtained which expresses a rare-cutting endonuclease able to selectivelyinactivate by DNA cleavage, preferably double-strand break, a geneencoding GCN2 and a rare-cutting endonuclease able to selectivelyinactivate by DNA cleavage, preferably double-strand break, a geneencoding PRDM1. According to particular embodiments, said immune cellcomprises one or more exogenous nucleic acid molecules comprising one ormore nucleotide sequences encoding said rare-cutting endonucleases.

According to certain embodiments, the engineered immune cell furtherexpresses a rare-cutting endonuclease able to selectively inactivate byDNA cleavage, preferably double-strand break, at least one gene codingfor a component of the T-cell receptor, such as TCR alpha. According toparticular embodiments, said immune cell comprises an exogenous nucleicacid molecule comprising a nucleotide sequence encoding saidrare-cutting endonuclease.

According to certain embodiments, the engineered immune cell furtherexpresses a Chimeric Antigen Receptor (CAR) directed against at leastone antigen expressed at the surface of a malignant cell. According toparticular embodiments, said immune cell comprises an exogenous nucleicacid molecule comprising a nucleotide sequence encoding said CAR.

It is understood that the details given herein in particularly withrespect to the rare-cutting endonuclease able to selectively inactivateby DNA cleavage the gene encoding GCN2, the rare-cutting endonucleaseable to selectively inactivate by DNA cleavage the gene encoding PRDM1,the rare-cutting endonuclease able to selectively inactivate by DNAcleavage at least one gene coding for a component of the T-cell receptor(TCR), and the Chimeric Antigen Receptor also apply to this aspect ofthe invention.

Further, in the scope of the present invention is also encompassed acell line obtained from an engineered immune cell according to theinvention.

As a result of the present invention, engineered immune cells can beused as therapeutic products, ideally as an “off the shelf” product, foruse in the treatment or prevention of medical conditions such as cancer.

Therapeutic Applications

Immune cells obtainable in accordance with the present invention areintended to be used as a medicament, and in particular for treatingcancer in a patient (e.g. a human patient) in need thereof. Accordingly,the present invention provides engineered immune cells for use as amedicament. Particularly, the present invention provides engineeredimmune cells for use in the treatment of a cancer. Also provided arecompositions, particularly pharmaceutical compositions, which compriseat least one engineered immune cell of the present invention. In certainembodiments, a composition may comprise a population of engineeredimmune cells of the present invention.

The treatment can be ameliorating, curative or prophylactic. It may beeither part of an autologous immunotherapy or part of an allogenicimmunotherapy treatment. By autologous, it is meant that cells, cellline or population of cells used for treating patients are originatingfrom said patient or from a Human Leucocyte Antigen (HLA) compatibledonor. By allogeneic is meant that the cells or population of cells usedfor treating patients are not originating from said patient but from adonor.

The invention is particularly suited for allogenic immunotherapy,insofar as it enables the transformation of immune cells, such asT-cells, typically obtained from donors, into non-alloreactive cells.This may be done under standard protocols and reproduced as many timesas needed. The resultant modified immune cells may be pooled andadministrated to one or several patients, being made available as an“off the shelf” therapeutic product.

The treatments are primarily to treat patients diagnosed with cancer.Particular cancers to be treated according to the invention are thosewhich have solid tumors, but may also concern liquid tumors. Adulttumors/cancers and pediatric tumors/cancers are also included.

According to certain embodiments, the engineered immune cell(s) orcomposition is for use in the treatment of a cancer, and moreparticularly for use in the treatment of a solid or liquid tumor.According to particular embodiments, the engineered immune cell(s) orcomposition is for use in the treatment of a solid tumor. According toother particular embodiments, the engineered immune cell(s) orcomposition is for use in the treatment of a liquid tumor.

According to particular embodiments, the engineered immune cell(s) orcomposition is for use in the treatment of a cancer selected from thegroup consisting of lung cancer, small lung cancer, breast cancer,uterine cancer, prostate cancer, kidney cancer, colon cancer, livercancer, pancreatic cancer, and skin cancer. According to more particularembodiments, the engineered immune cell(s) or composition is for use inthe treatment of lung cancer. According to other more particularembodiments, the engineered immune cell(s) or composition is for use inthe treatment of small lung cancer. According to other more particularembodiments, the engineered immune cell(s) or composition is for use inthe treatment of breast cancer. According to other more particularembodiments, the engineered immune cell(s) or composition is for use inthe treatment of uterine cancer. According to other more particularembodiments, the engineered immune cell(s) or composition is for use inthe treatment of prostate cancer. According to other more particularembodiments, the engineered immune cell(s) or composition is for use inthe treatment of kidney cancer. According to other more particularembodiments, the engineered immune cell(s) or composition is for use inthe treatment of colon cancer. According to other more particularembodiments, the engineered immune cell(s) or composition is for use inthe treatment of liver cancer. According to other more particularembodiments, the engineered immune cell(s) or composition is for use inthe treatment of pancreatic cancer. According to other more particularembodiments, the engineered immune cell(s) or composition is for use inthe treatment of skin cancer.

According to other particular embodiments, the engineered immune cell(s)or composition is for use in the treatment of a sarcoma.

According to other particular embodiments, the engineered immune cell(s)or composition is for use in the treatment of a carcinoma. According tomore particular embodiments, the engineered immune cell or compositionis for use in the treatment of renal, lung or colon carcinoma.

According to other particular embodiments, the engineered immune cell(s)or composition is for use in the treatment of leukemia, such as acutelymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chroniclymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), andchronic myelomonocystic leukemia (CMML). According to more particularembodiments, the engineered immune cell(s) or composition is for use inthe treatment of acute lymphoblastic leukemia (ALL). According to othermore particular embodiments, the engineered immune cell(s) orcomposition is for use in the treatment of acute myeloid leukemia (AML).According to other more particular embodiments, the engineered immunecell(s) or composition is for use in the treatment of chroniclymphocytic leukemia (CLL). According to other more particularembodiments, the engineered immune cell(s) or composition is for use inthe treatment of chronic myelogenous leukemia (CML). According to othermore particular embodiments, the engineered immune cell(s) orcomposition is for use in the treatment of chronic myelomonocysticleukemia (CMML).

According to other particular embodiments, the engineered immune cell(s)or composition is for use in the treatment of lymphoma, such as B-celllymphoma. According to more particular embodiments, the engineeredimmune cell(s) or composition is for use in the treatment of primary CNSlymphoma. According to other more particular embodiments, the engineeredimmune cell(s) or composition is for use in the treatment of Hodgkin'slymphoma. According to other more particular embodiments, the engineeredimmune cell(s) or composition is for use in the treatment ofNon-Hodgkin's lymphoma. According to more particular embodiments, theengineered immune cell(s) or composition is for use in the treatment ofdiffuse large B cell lymphoma (DLBCL). According to other moreparticular embodiments, the engineered immune cell(s) or composition isfor use in the treatment of Follicular lymphoma. According to other moreparticular embodiments, the engineered immune cell(s) or composition isfor use in the treatment of marginal zone lymphoma (MZL). According toother more particular embodiments, the engineered immune cell(s) orcomposition is for use in the treatment of Mucosa-Associated LymphaticTissue lymphoma (MALT). According to other more particular embodiments,the engineered immune cell(s) or composition is for use in the treatmentof small cell lymphocytic lymphoma. According to other more particularembodiments, the engineered immune cell(s) or composition is for use inthe treatment of mantle cell lymphoma (MCL). According to other moreparticular embodiments, the engineered immune cell(s) or composition isfor use in the treatment of Burkitt lymphoma. According to other moreparticular embodiments, the engineered immune cell(s) or composition isfor use in the treatment of primary mediastinal (thymic) large B-celllymphoma. According to other more particular embodiments, the engineeredimmune cell(s) or composition is for use in the treatment of Waldenströmmacroglobulinemia. According to other more particular embodiments, theengineered immune cell(s) or composition is for use in the treatment ofnodal marginal zone B cell lymphoma (NMZL). According to other moreparticular embodiments, the engineered immune cell(s) or composition isfor use in the treatment of splenic marginal zone lymphoma (SMZL).According to other more particular embodiments, the engineered immunecell(s) or composition is for use in the treatment of intravascularlarge B-cell lymphoma. According to other more particular embodiments,the engineered immune cell(s) or composition is for use in the treatmentof Primary effusion lymphoma. According to other more particularembodiments, the engineered immune cell(s) or composition is for use inthe treatment of lymphomatoid granulomatosis. According to other moreparticular embodiments, the engineered immune cell(s) or composition isfor use in the treatment of T cell/histiocyte-rich large B-celllymphoma. According to other more particular embodiments, the engineeredimmune cell(s) or composition is for use in the treatment of primarydiffuse large B-cell lymphoma of the CNS (Central Nervous System).According to other more particular embodiments, the engineered immunecell(s) or composition is for use in the treatment of primary cutaneousdiffuse large B-cell lymphoma. According to other more particularembodiments, the engineered immune cell(s) or composition is for use inthe treatment of EBV positive diffuse large B-cell lymphoma of theelderly. According to other more particular embodiments, the engineeredimmune cell(s) or composition is for use in the treatment of diffuselarge B-cell lymphoma associated with inflammation. According to othermore particular embodiments, the engineered immune cell(s) orcomposition is for use in the treatment of ALK-positive large B-celllymphoma. According to other more particular embodiments, the engineeredimmune cell(s) or composition is for use in the treatment ofplasmablastic lymphoma. According to other more particular embodiments,the engineered immune cell(s) or composition is for use in the treatmentof Large B-cell lymphoma arising in HHV8-associated multicentricCastleman disease.

According to certain embodiment, the engineered immune cell originatesfrom a patient, e.g. a human patient, to be treated. According tocertain other embodiment, the engineered immune cell originates from atleast one donor.

The treatment can take place in combination with one or more therapiesselected from the group of antibodies therapy, chemotherapy, cytokinestherapy, dendritic cell therapy, gene therapy, hormone therapy, laserlight therapy and radiation therapy.

According to certain embodiments, immune cells of the invention canundergo robust in vivo immune cell expansion upon administration to apatient, and can persist in the body fluids for an extended amount oftime, preferably for a week, more preferably for 2 weeks, even morepreferably for at least one month. Although the immune cells accordingto the invention are expected to persist during these periods, theirlife span into the patient's body are intended not to exceed a year,preferably 6 months, more preferably 2 months, and even more preferablyone month.

The administration of the cells or population of cells according to thepresent invention may be carried out in any convenient manner, includingby aerosol inhalation, injection, ingestion, transfusion, implantationor transplantation. The compositions described herein may beadministered to a patient subcutaneously, intradermaliy, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous orintralymphatic injection, or intraperitoneally. In one embodiment, thecell compositions of the present invention are preferably administeredby intravenous injection.

The administration of the cells or population of cells can consist ofthe administration of 104-109 cells per kg body weight, preferably 105to 106 cells/kg body weight including all integer values of cell numberswithin those ranges. The cells or population of cells can beadministrated in one or more doses. In another embodiment, saideffective amount of cells are administrated as a single dose. In anotherembodiment, said effective amount of cells are administrated as morethan one dose over a period time. Timing of administration is within thejudgment of managing physician and depends on the clinical condition ofthe patient. The cells or population of cells may be obtained from anysource, such as a blood bank or a donor. While individual needs vary,determination of optimal ranges of effective amounts of a given celltype for a particular disease or conditions within the skill of the art.An effective amount means an amount which provides a therapeutic orprophylactic benefit. The dosage administrated will be dependent uponthe age, health and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment and the nature of the effectdesired.

In other embodiments, said effective amount of cells or compositioncomprising those cells are administrated parenterally. Saidadministration can be an intravenous administration. Said administrationcan be directly done by injection within a tumor.

In certain embodiments, cells are administered to a patient inconjunction with (e.g., before, simultaneously or following) any numberof relevant treatment modalities, including but not limited to treatmentwith agents such as antiviral therapy, cidofovir and interleukin-2,Cytarabine (also known as ARA-C) or nataliziimab treatment for MSpatients or efaliztimab treatment for psoriasis patients or othertreatments for PML patients. In further embodiments, the T cells of theinvention may be used in combination with chemotherapy, radiation,immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAMPATH, anti-CD3 antibodies or otherantibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin,mycoplienolic acid, steroids, FR901228, cytokines, and irradiation.These drugs inhibit either the calcium dependent phosphatase calcineurin(cyclosporine and FK506) or inhibit the p70S6 kinase that is importantfor growth factor induced signaling (rapamycin) (Liu et al., Cell66:807-815, 11; Henderson et al., Immun. 73:316-321, 1991; Bierer etal., Citrr. Opin. mm n. 5:763-773, 93). In a further embodiment, thecell compositions of the present invention are administered to a patientin conjunction with (e.g., before, simultaneously or following) bonemarrow transplantation, T cell ablative therapy using eitherchemotherapy agents such as, fludarabine, external-beam radiationtherapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH,In another embodiment, the cell compositions of the present inventionare administered following B-cell ablative therapy such as agents thatreact with CD20, e.g., Rituxan. For example, in one embodiment, subjectsmay undergo standard treatment with high dose chemotherapy followed byperipheral blood stem cell transplantation. In certain embodiments,following the transplant, subjects receive an infusion of the expandedgenetically engineered immune cells of the present invention. In anadditional embodiment, expanded cells are administered before orfollowing surgery.

Also encompassed within this aspect of the invention are methods fortreating a patient in need thereof, comprising a) providing at least oneengineered immune cell of the present invention, preferably a populationof said immune cell; and b) administering said immune cell or populationto said patient.

Also encompassed within this aspect of the invention are methods forpreparing a medicament using at least one engineered immune cell of thepresent invention, and preferably a population of said immune cell.Accordingly, the present invention provides the use of at least oneengineered immune cell of the present invention, and preferably apopulation of said immune cell, in the manufacture of a medicament.Preferably, such medicament is for use in the treatment of a disease asspecified above.

OTHER DEFINITIONS

-   -   Amino acid residues in a polypeptide sequence are designated        herein according to the one-letter code, in which, for example,        Q means Gln or Glutamine residue, R means Arg or Arginine        residue and D means Asp or Aspartic acid residue.    -   Amino acid substitution means the replacement of one amino acid        residue with another, for instance the replacement of an        Arginine residue with a Glutamine residue in a peptide sequence        is an amino acid substitution.    -   Nucleotides are designated as follows: one-letter code is used        for designating the base of a nucleoside: a is adenine, t is        thymine, c is cytosine, and g is guanine. For the degenerated        nucleotides, r represents g or a (purine nucleotides), k        represents g or t, s represents g or c, w represents a or t, m        represents a or c, y represents t or c (pyrimidine nucleotides),        d represents g, a or t, v represents g, a or c, b represents g,        t or c, h represents a, t or c, and n represents g, a, t or c.    -   “As used herein, “nucleic acid” or “polynucleotides” refers to        nucleotides and/or polynucleotides, such as deoxyribonucleic        acid (DNA) or ribonucleic acid (RNA), oligonucleotides,        fragments generated by the polymerase chain reaction (PCR), and        fragments generated by any of ligation, scission, endonuclease        action, and exonuclease action. Nucleic acid molecules can be        composed of monomers that are naturally-occurring nucleotides        (such as DNA and RNA), or analogs of naturally-occurring        nucleotides (e.g., enantiomeric forms of naturally-occurring        nucleotides), or a combination of both. Modified nucleotides can        have alterations in sugar moieties and/or in pyrimidine or        purine base moieties. Sugar modifications include, for example,        replacement of one or more hydroxyl groups with halogens, alkyl        groups, amines, and azido groups, or sugars can be        functionalized as ethers or esters. Moreover, the entire sugar        moiety can be replaced with sterically and electronically        similar structures, such as aza-sugars and carbocyclic sugar        analogs. Examples of modifications in a base moiety include        alkylated purines and pyrimidines, acylated purines or        pyrimidines, or other well-known heterocyclic substitutes.        Nucleic acid monomers can be linked by phosphodiester bonds or        analogs of such linkages. Nucleic acids can be either single        stranded or double stranded.    -   by “DNA target”, “DNA target sequence”, “target DNA sequence”,        “nucleic acid target sequence”, “target sequence”, or        “processing site” is intended a polynucleotide sequence that can        be targeted and processed by a rare-cutting endonuclease        according to the present invention. These terms refer to a        specific DNA location, preferably a genomic location in a cell,        but also a portion of genetic material that can exist        independently to the main body of genetic material such as        plasmids, episomes, virus, transposons or in organelles such as        mitochondria as non-limiting example. As non-limiting examples        of RNA guided target sequences, are those genome sequences that        can hybridize the guide RNA which directs the RNA guided        endonuclease to a desired locus.    -   By “delivery vector” or “delivery vectors” is intended any        delivery vector which can be used in the present invention to        put into cell contact (i.e “contacting”) or deliver inside cells        or subcellular compartments (i.e “introducing”) agents/chemicals        and molecules (proteins or nucleic acids) needed in the present        invention. It includes, but is not limited to liposomal delivery        vectors, viral delivery vectors, drug delivery vectors, chemical        carriers, polymeric carriers, lipoplexes, polyplexes,        dendrimers, microbubbles (ultrasound contrast agents),        nanoparticles, emulsions or other appropriate transfer vectors.        These delivery vectors allow delivery of molecules, chemicals,        macromolecules (genes, proteins), or other vectors such as        plasmids, or penetrating peptides. In these later cases,        delivery vectors are molecule carriers.    -   The terms “vector” or “vectors” refer to a nucleic acid molecule        capable of transporting another nucleic acid to which it has        been linked. A “vector” in the present invention includes, but        is not limited to, a viral vector, a plasmid, a RNA vector or a        linear or circular DNA or RNA molecule which may consists of a        chromosomal, non-chromosomal, semi-synthetic or synthetic        nucleic acids. Preferred vectors are those capable of autonomous        replication (episomal vector) and/or expression of nucleic acids        to which they are linked (expression vectors). Large numbers of        suitable vectors are known to those of skill in the art and        commercially available.

Viral vectors include retrovirus, adenovirus, parvovirus (e. g.adenoassociated viruses), coronavirus, negative strand RNA viruses suchas orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies andvesicular stomatitis virus), paramyxovirus (e. g. measles and Sendai),positive strand RNA viruses such as picornavirus and alphavirus, anddouble-stranded DNA viruses including adenovirus, herpesvirus (e.g.,Herpes Simplex virus types 1 and 2, Epstein-Barr virus,cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox).Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses,papovavirus, hepadnavirus, and hepatitis virus, for example. Examples ofretroviruses include: avian leukosis-sarcoma, mammalian C-type, B-typeviruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin,J. M., Retroviridae: The viruses and their replication, In FundamentalVirology, Third Edition, B. N. Fields, et al., Eds., Lippincott-RavenPublishers, Philadelphia, 1996).

-   -   By “lentiviral vector” is meant HIV-Based lentiviral vectors        that are very promising for gene delivery because of their        relatively large packaging capacity, reduced immunogenicity and        their ability to stably transduce with high efficiency a large        range of different cell types. Lentiviral vectors are usually        generated following transient transfection of three (packaging,        envelope and transfer) or more plasmids into producer cells.        Like HIV, lentiviral vectors enter the target cell through the        interaction of viral surface glycoproteins with receptors on the        cell surface. On entry, the viral RNA undergoes reverse        transcription, which is mediated by the viral reverse        transcriptase complex. The product of reverse transcription is a        double-stranded linear viral DNA, which is the substrate for        viral integration in the DNA of infected cells. By “integrative        lentiviral vectors (or LV)”, is meant such vectors as non        limiting example, that are able to integrate the genome of a        target cell. At the opposite by “non integrative lentiviral        vectors (or NILV)” is meant efficient gene delivery vectors that        do not integrate the genome of a target cell through the action        of the virus integrase.    -   Delivery vectors and vectors can be associated or combined with        any cellular permeabilization techniques such as sonoporation or        electroporation or derivatives of these techniques.    -   By “cell” or “cells” is intended any eukaryotic living cells,        primary cells and cell lines derived from these organisms for in        vitro cultures.    -   By “primary cell” or “primary cells” are intended cells taken        directly from living tissue (i.e. biopsy material) and        established for growth in vitro, that have undergone very few        population doublings and are therefore more representative of        the main functional components and characteristics of tissues        from which they are derived from, in comparison to continuous        tumorigenic or artificially immortalized cell lines.

As non-limiting examples cell lines can be selected from the groupconsisting of CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells,U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLacells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; Molt 4cells.

All these cell lines can be modified by the method of the presentinvention to provide cell line models to produce, express, quantify,detect, study a gene or a protein of interest; these models can also beused to screen biologically active molecules of interest in research andproduction and various fields such as chemical, biofuels, therapeuticsand agronomy as non-limiting examples.

-   -   By “stem cell” is meant a cell that has the capacity to        self-renew and the ability to generate differentiated cells.        More explicitly, a stem cell is a cell which can generate        daughter cells identical to their mother cell (self-renewal) and        can produce progeny with more restricted potential        (differentiated cells).    -   By “NK cells” is meant natural killer cells. NK cells are        defined as large granular lymphocytes and constitute the third        kind of cells differentiated from the common lymphoid progenitor        generating B and T lymphocytes.    -   by “mutation” is intended the substitution, deletion, insertion        of up to one, two, three, four, five, six, seven, eight, nine,        ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty        five, thirty, forty, fifty, or more nucleotides/amino acids in a        polynucleotide (cDNA, gene) or a polypeptide sequence. The        mutation can affect the coding sequence of a gene or its        regulatory sequence. It may also affect the structure of the        genomic sequence or the structure/stability of the encoded mRNA.    -   by “variant(s)”, it is intended a repeat variant, a variant, a        DNA binding variant, a TALE-nuclease variant, a polypeptide        variant obtained by mutation or replacement of at least one        residue in the amino acid sequence of the parent molecule.    -   by “functional variant” is intended a catalytically active        mutant of a protein or a protein domain; such mutant may have        the same activity compared to its parent protein or protein        domain or additional properties, or higher or lower activity.    -   By “gene” is meant the basic unit of heredity, consisting of a        segment of DNA arranged in a linear manner along a chromosome,        which codes for a specific protein or segment of protein. A gene        typically includes a promoter, a 5′ untranslated region, one or        more coding sequences (exons), optionally introns, a 3′        untranslated region. The gene may further comprise a terminator,        enhancers and/or silencers.    -   As used herein, the term “locus” is the specific physical        location of a DNA sequence (e.g. of a gene) on a chromosome. The        term “locus” can refer to the specific physical location of a        rare-cutting endonuclease target sequence on a chromosome. Such        a locus can comprise a target sequence that is recognized and/or        cleaved by a rare-cutting endonuclease according to the        invention. It is understood that the locus of interest of the        present invention can not only qualify a nucleic acid sequence        that exists in the main body of genetic material (i.e. in a        chromosome) of a cell but also a portion of genetic material        that can exist independently to said main body of genetic        material such as plasmids, episomes, virus, transposons or in        organelles such as mitochondria as non-limiting examples.    -   The term “cleavage” refers to the breakage of the covalent        backbone of a polynucleotide. Cleavage can be initiated by a        variety of methods including, but not limited to, enzymatic or        chemical hydrolysis of a phosphodiester bond. Both        single-stranded cleavage and double-stranded cleavage are        possible, and double-stranded cleavage can occur as a result of        two distinct single-stranded cleavage events. Double stranded        DNA, RNA, or DNA/RNA hybrid cleavage can result in the        production of either blunt ends or staggered ends.    -   By “fusion protein” is intended the result of a well-known        process in the art consisting in the joining of two or more        genes which originally encode for separate proteins or part of        them, the translation of said “fusion gene” resulting in a        single polypeptide with functional properties derived from each        of the original proteins.    -   “identity” refers to sequence identity between two nucleic acid        molecules or polypeptides. Identity can be determined by        comparing a position in each sequence which may be aligned for        purposes of comparison. When a position in the compared sequence        is occupied by the same base or amino acid, then the molecules        are identical at that position. A degree of similarity or        identity between nucleic acid or amino acid sequences is a        function of the number of identical or matching nucleotides or        amino acids at positions shared by the nucleic acid or amino        acid sequences, respectively. Various alignment algorithms        and/or programs may be used to calculate the identity between        two sequences, including FASTA, or BLAST which are available as        a part of the GCG sequence analysis package (University of        Wisconsin, Madison, Wis.), and can be used with, e.g., default        setting. For example, polypeptides having at least 70%, 85%,        90%, 95%, 98% or 99% identity to specific polypeptides described        herein and preferably exhibiting substantially the same        functions, as well as polynucleotide encoding such polypeptides,        are contemplated.    -   “co-stimulatory ligand” refers to a molecule on an antigen        presenting cell that specifically binds a cognate co-stimulatory        molecule on a T-cell, thereby providing a signal which, in        addition to the primary signal provided by, for instance,        binding of a TCR/CD3 complex with an MHC molecule loaded with        peptide, mediates a T cell response, including, but not limited        to, proliferation activation, differentiation and the like. A        co-stimulatory ligand can include but is not limited to CD7,        B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible        costimulatory igand (ICOS-L), intercellular adhesion molecule        (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM,        lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, an agonist or        antibody that binds Toll ligand receptor and a ligand that        specifically binds with B7-H3. A co-stimulatory ligand also        encompasses, inter alia, an antibody that specifically binds        with a co-stimulatory molecule present on a T cell, such as but        not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS,        lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7,        LTGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.    -   A “co-stimulatory molecule” refers to the cognate binding        partner on a Tcell that specifically binds with a co-stimulatory        ligand, thereby mediating a co-stimulatory response by the cell,        such as, but not limited to proliferation. Co-stimulatory        molecules include, but are not limited to an MHC class I        molecule, BTLA and Toll ligand receptor.    -   A “co-stimulatory signal” as used herein refers to a signal,        which in combination with primary signal, such as TCR/CD3        ligation, leads to T cell proliferation and/or upregulation or        downregulation of key molecules.    -   The term “extracellular ligand-binding domain” as used herein is        defined as an oligo- or polypeptide that is capable of binding a        ligand. Preferably, the domain will be capable of interacting        with a cell surface molecule. For example, the extracellular        ligand-binding domain may be chosen to recognize a ligand that        acts as a cell surface marker on target cells associated with a        particular disease state. Thus examples of cell surface markers        that may act as ligands include those associated with viral,        bacterial and parasitic infections, autoimmune disease and        cancer cells.    -   The term “subject” or “patient” as used herein includes all        members of the animal kingdom including non-human primates and        humans.    -   The above written description of the invention provides a manner        and process of making and using it such that any person skilled        in this art is enabled to make and use the same, this enablement        being provided in particular for the subject matter of the        appended claims, which make up a part of the original        description.

Where a numerical limit or range is stated herein, the endpoints areincluded. Also, all values and sub ranges within a numerical limit orrange are specifically included as if explicitly written out.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples, which areprovided herein for purposes of illustration only, and are not intendedto be limiting unless otherwise specified.

EXAMPLES Example 1: T-Cell Sensitivity to Arginase Activity

To verify that T-cells were sensitive to arginine deprivation byarginase I activity in their microenvironment, Xcico15 mediacomplemented with 5% human AB serum and 20 ng/ml human IL2 (100 μl perwell in a 96-well plate) was incubated with increasing concentrations ofrecombinant arginase I (0.5 to 1500 ng/μl).

After 3 days at 4° C., human T-cells that had previously beentransfected (PulseAgile) with mRNA encoding a TRAC specific TALEnuclease or no RNA were resuspended in the arginase-treated media. After72 hours at 37° C., cell viability was measured by flow cytometry. Theresults are depicted in FIG. 2.

As can be seen from FIG. 2, increasing concentrations of arginase, andthus decreasing concentrations of arginine in the media leads to adrastic decrease in viable T-cells. These results suggest that bothT-cells treated with TRAC specific TALE nuclease (KO TRAC) and untreatedT-cells (WT) are sensitive to arginine deprivation in theirmicroenvironment in vitro.

Example 2: GCN2 Disruption by Use of TALE Nucleases

Two TALE nucleases (GCN2_1 and GCN2_2) were designed to disrupt the GCN2gene in human T-cells. mRNA encoding TALE nucleases targeting the humanGCN2 gene were ordered from Cellectis Bioresearch (8, rue de la CroixJarry, 75013 Paris). Table 2 below indicates the target sequence cleavedby the respective TALE nuclease.

TABLE 2  TALE nucleases targeting human GCN2 gene target sequence GCN2_1TGGATTTGAGGGTTAAATGCCCACCTACCTATCCAGAT GTGTGAGTACA (SEQ ID NO: 3) GCN2_2TTGTAGGAAATGGTAAACATCGGGCAAACTCCTCAGGA AGGTCTAGGTA (SEQ ID NO: 4)

Human T-cells were transfected with mRNA encoding either of said TALEnucleases. Control cells were transfected without RNA. 3 days posttransfection genomic DNA was isolated and subjected to T7 endonucleaseassay to detect TALE nuclease activity. The results are depicted in FIG.3.

As can be seen from FIG. 3, the presence of lower molecular bandscompared to the sample without RNA transfection clearly indicatedcleavage activity of both TALE nucleases.

To test whether GCN2 disruption conferred resistance to argininedeprivation by arginase, TALEN treated T cells as well as control cellswere incubated in RPMI1640 medium prepared without arginine wherearginine was added in increasing concentration. After 72 h incubation at37° C., cell viability was measured by flow cytometry.

As can be seen from FIG. 4, T-cells treated with GCN2 TALEN survivedbetter at lower concentrations of arginine. This suggests that immunecells, and especially T-cells, having a disrupted GCN2 gene, and thus donot express the GCN2 protein in a functional form, provide resistance toimmunosuppression in a tumor microenvironment where arginase issecreted.

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The invention claimed is:
 1. A method of treating an arginase Iproducing cancer that inhibits T-cell activity, comprising administeringto a patient in need thereof a therapeutically effective amount of anengineered human T-cell comprising at least one insertion, deletion, oraddition to a GCN2 or PRDM1 gene conferring arginase resistance, whereinsaid cancer is selected from the group consisting of lung cancer, breastcancer, uterine cancer, prostate cancer, kidney cancer, colon cancer,liver cancer, pancreatic cancer, and skin cancer.
 2. The methodaccording to claim 1, wherein the GCN2 gene or PRDM1 gene is inactivatedby cleavage with a rare-cutting endonuclease.
 3. The method according toclaim 1, wherein the T-cell is an autologous T-cell.
 4. The methodaccording to claim 1, wherein the T-cell is from a donor.